Mitomycin biosynthetic gene cluster

ABSTRACT

The invention provides a biosynthetic gene cluster for mitomycin, as well as methods of using gene(s) within the cluster to alter biosynthesis.

BACKGROUND OF THE INVENTION

[0001] Streptomyces are filamentous Gram-positive soil bacteria with a nucleotide base composition greater than 70 mole % G+C (Stackebrandt and Woese, 1981). They produce a wide array of biologically active compounds including over two thirds of the commercially important natural product metabolites (Alderson et al., 1993; Bevax, 1998). Genetic information accumulated over the past 15 years has demonstrated that genes encoding enzymes for natural product assembly are clustered on the Streptomyces genome (Martin, 1992). In addition, one or more pathway-specific transcriptional regulatory genes, and at least one resistance gene are typically found within the antibiotic biosynthetic gene cluster (Chater, 1992). Heterologous hybridization with gene probes based on highly conserved biosynthetic enzyme amino acid sequences has been useful to clone antibiotic biosynthetic genes (Hopwood, 1997; Seno and Baltz, 1989; Turgay and Marahiel, 1994).

[0002] The mitomycins are a group of natural products that contain a variety of functional groups, including aminobenzoquinone and aziridine ring systems. One representative of the family, mitomycin C (MC), was the first recognized bioreductive alkylating agent. In particular, since its discovery and demonstration of anticancer activity in the 1960s, many aspects of the chemistry and biology of MC have been investigated. This has provided detailed information on its unprecedented molecular mechanism, unique biological and pharmacological properties, drug resistance, and bioactive analogues (Hata et al., 1956; Verweij, 1997). MC is regarded as the prototype natural product alkylating agent whose activity is dependent on the reductive activation (either chemically, such as low pH, or enzymatically, such as DT-diaphorase, NADH cytochrome c reductase) (Boxer, 1997; Cummings et al., 1998). Activated MC crosslinks double-stranded DNA, which in turn induces diverse biological effects including selective inhibition of DNA synthesis, mutagenesis, induction of DNA repair (SOS response), sister chromatid exchange, signal transduction, and induction of apoptosis (Tomasz and Palem, 1997). Tumor hypoxia and the increased expression of bioreductive enzymes in malignant cells create a selective environment for drug activation and make MC an attractive agent for anti-tumor therapy (Spanswick et al., 1998). MC has become one of the most effective antitumor drugs against non-small cell lung carcinoma and other soft tumors, as well as a clinically important component of combination cancer chemotherapy and radiotherapy of solid tumors (Henderson, 1993).

[0003] In addition to its biological and pharmacological importance, MC is prominent because its molecular mechanism represents a model for structurally related antitumor antibiotics such as porfiromycin (Pan and Iracki, 1988), mitiromycin (Wakaki et al., 1958), FR66979 (Paz and Hopkins, 1997), FR900482 (Williams et al., 1997), FK973 (Hirai et al., 1994), and FK317 (Naoe et al., 1998), as well as structurally unrelated bioreductive agents such as EO9 (Smitskampwilms et al., 1996), and tirapazamine (Evans et al., 1998). Numerous MC derivatives have been synthesized and tested for enhanced activities, including the recently identified selective protein tyrosine kinase inhibitor, la-docosahexaenoyl MC (Kasai and Arai, 1995; Shikano et al., 1998).

[0004]Streptomyces lavendulae produces MC. The molecule has an unusual structure comprised of aziridine, pyrrolizidine, pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give the mitosane nucleus (Webb et al., 1962). The mitosane core of MC was shown to be derived from the junction of an arnino-methylbenzoquinone (mC₇N unit) and hexosamine (C₆N unit) (Homemann, 1981). The C₆N unit consists of carbons 1, 2, 3, 9, 9a, 10, with the aziridine nitrogen derived intact from D-glucosamine (Hornemann et al., 1974).

[0005] The mC₇N unit in MC and the ansamycins is derived from 3-amino-5-hydroxybenzoic acid (AHBA) (Becker et al., 1983; Kibby and Richards, 1981). AHBA was first shown to be incorporated into the ansamycin antibiotic actamycin (Kibby et al., 1980). Subsequently, it was confirmed as an efficient precursor for rifamycin (Becker et al., 1983; Kibby and Rickards, 1981; Ghilsalba and Neuesch, 1981), geldanamycin (Potgieter, 1983), ansamitocin (Hatano et al., 1982), ansatrienin (Wu et al., 1987), streptovaricin (Staley and Rinehart, 1991) and naphthomycin A (Lee et al., 1994). Anderson et al. (1980) demonstrated that [carboxy-¹³C] AHBA could be efficiently and specifically incorporated into the C-6 methyl group of porfiromycin, which contains the same mitosane core as MC. Incorporation experiments with radiolabeled precursors have demonstrated that the mitosane core of MC was derived from the junction of AHBA and D-glucosamine (Anderson et al., 1980; Hornemann, 1981).

[0006] Meanwhile the O- and N- (but not C-) methyl groups were shown to be derived from L-methionine, while the C-10 carbamoyl group came from L-arginine or L-citrulline (Bezanson and Vining, 1971; Hornemann and Eggert, 1975; Homemann et al., 1974). [¹⁴C]-labeled precursor feeding studies with D-glucose, pyruvate and D-erytrose indicated that de novo biosynthesis of AHBA resulted directly from the shildiate pathway. However, no incorporation into the mC₇N unit of either MC (Hornemann, 1981) or the ansamycin antibiotics (Chiao et al., 1998) was found from labeling studies with shikimic acid, the shikimate precursor 3-dehydroshikimic acid, or the shikdmate derived amino acids. These results led to the hypothesis of a modified shikimate pathway, in which a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase-like enzyme catalyzes the conversion to 3,4-dideoxy4-amino-D-arabino-heptulosonic acid-7-phosphate (amino-DABP), to give the ammoniated shikimate pathway (Kim et al., 1992). Floss (1997) provided strong support for this new variant of the shikimate pathway by showing that aminoDAHP, 5-deoxy-5-amino-3dehydroshikimic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be efficiently converted into AHBA by a cell-free extract of Amycolatopsis mediteranei (rifamycin producer), in contrast to the normal shikimate pathway intermediate DAHP which was not converted (Kim et al., 1992; Kim et al., 1996). Recently, the AHBA synthase (rifK) gene from A. mediteranei has been cloned, sequenced and functionally characterized (Kim et al., 1998).

[0007] Little is known regarding the details of the convergent assembly of MC from AHBA and D-glucosamine in S. lavendulae, i.e., whether its de novo biosynthesis is related to the primary metabolic shikimate pathway, an important route in microorganisms and plants for aromatic amino acid biosynthesis (Floss, 1997). In addition, it is unclear how S. lavendulae resists the activity of MC since the preferred MC alkylation sites in DNA are guanine and cytosine, and MC-induced cell death can result from a single crosslink per genome (Tomasz, 1995).

[0008] Thus, there is a continuing need for the identification and isolation of antibiotic biosynthetic genes, including genes which confer resistance to antibiotics or result in enhanced production of antibiotics.

SUMMARY OF THE INVENTION

[0009] The present invention provides an isolated and purified nucleic acid molecule, e.g., DNA, comprising a gene cluster for mitomycin, a variant or a fragment thereof (the mit/mmc gene cluster). As described hereinbelow, the S. lavendulae mitomycin gene cluster includes the mitomycin biosynthetic gene cluster comprising 47 mitomycin biosynthetic genes spanning 55 kb of contiguous DNA. The biosynthetic portion of the gene cluster includes genes that encode polypeptides involved in the generation of biosynthetic precursors, mitosane ring system assembly and functionalization (e.g., methylation, hydroxylation, aminotransfer, carbamoylation, and carbonyl reduction), a mitomycin resistance gene which is different than mrd and the unlinked mcr, as well as several regulatory genes. Gene disruption was employed to further characterize some of the genes. Fourteen of 22 gene disruption mutants affected mitomycin biosynthesis, resulting in abrogation or overexpression of drug production, e.g., targeted genetic disruption of a mitomycin pathway regulator (e.g., mmcW) led to a substantial increase in drug production. It is preferred that the isolated and purified nucleic acid molecule of the invention is nucleic acid from Streptomyces spp., such as Streptomyces lavendulae (e.g., B19/ATCC 27422, NRRL 2564, KY68 1, ATCC 27423, or PB 1000), Streptomyces caespitosus, Streptomyces verticillatus, and Streptomyces sandaensis (FERM-P7654), although isolated and purified nucleic acid molecules from other organisms which produce mitomycin or biological or functional equivalents thereof are also within the scope of the invention. The nucleic acid molecules of the invention are double-stranded or single-stranded.

[0010] As described hereinbelow, a 3.8 kb BamHI fragment from the S. lavendulae genome was isolated which comprises three open reading frames (ORFs). One of the ORFs (mitA) showed high similarity to previously identified AHBA synthase genes (Kim et al., 1998), while another (mitB) showed sequence similarity to several prokaryotic and eukaryotic glycosyltransferases. Nucleotide sequence analysis showed that mitA encodes a 388 amino acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediteranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of a S. lavendulae strain containing a K191A mutation in MitA with 3-amino-5-hydroxybenzoic acid, i.e., MC production was restored when the mitA mutant strain was cultured in the presence of exogenous 3-amino-5-hydroxybenzoic acid. mitB encodes a 272 amino acid protein.

[0011] Seven gene products (aminoDHQ synthase (MitP), aminoquinate dehydrogenase (MitT), aminoDHQ dehydratase (MmcF), AHBA synthase (MitA), oxidoreductase (MitG), phosphatase (MitJ), and kinase (MitS)) are likely responsible for assembly of the intermediate 3-amino-5-hydroxybenzoic acid (AHBA) through a variant of the shikimate pathway. However, the gene encoding aminoDAHP synthase, the first presumed enzyme involved in AHBA biosynthesis from phosphoenol pyruvate (PEP) and erythrose 4-phosphate (E4P), is not linked within the mitomycin biosynthetic gene cluster.

[0012] A mitomycin resistance determinant (mct) encodes a membrane-associated protein involved in excretion of mitomycin from cells. Disruption of met by insertional inactivation resulted in a S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in E. coli conferred a 5-fold increase in cellular resistance to MC, led to the synthesis of a membrane associated protein, and correlated with reduced intracellular accumulation of drug. Co-expression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. The results establish that MRD maintains a high affinity for MC and may serve as the primary receptor (participating as an accessory component in a drug export system) for subsequent transport by MCT.

[0013] The cloned mitomycin biosynthetic genes are useful to elucidate the molecular basis for the biosynthesis of the mitosane ring system, as well as to engineer the biosynthesis of novel natural products. Moreover, genetic engineering or overexpression of the transport, resistance and regulatory proteins may lead to higher titers of mitomycin compounds from production cultures.

[0014] Preferably, the isolated nucleic acid molecule comprising the gene cluster includes a nucleic acid sequence comprising SEQ ID NO:96 or SEQ ID NO:76, a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:96, SEQ ID NO:76, the complement thereof, or a fragment thereof. Moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). For example, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50° C., or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

[0015] A preferred nucleic molecule of the invention comprises a nucleic acid sequence encoding a polypeptide including, but not limited to, MitA (e.g., SEQ ID NO:10 encoded by SEQ ID NO:97), MitB (e.g., SEQ ID NO:11 encoded by SEQ ID NO:98), MitC (e.g., SEQ ID NO:12 encoded by SEQ ID NO:99), MitD (e.g., SEQ ID NO:100 encoded by SEQ ID NO:45), MitE (e.g., SEQ ID NO:101 encoded by SEQ ID NO:44), MitF (e.g., SEQ ID NO:102 encoded by SEQ ID NO:43), MitG (e.g., SEQ ID NO:103 encoded by SEQ ID NO:42), MitH (e.g., SEQ ID NO:104 encoded by SEQ ID NO:41), MitI (e.g., SEQ ID NO:105 encoded by SEQ ID NO:40), MitJ (e.g., SEQ ID NO:106 encoded by SEQ ID NO:39), MitK (e.g., SEQ ID NO:107 encoded by SEQ ID NO:38), MitL (e.g., SEQ ID NO:108 encoded by SEQ ID NO:37), MitM (e.g., SEQ ID NO:109 encoded by SEQ ID NO:36), MitN (e.g., SEQ ID NO:108 encoded by SEQ ID NO:35), MitO (e.g., SEQ ID NO:111 encoded by SEQ ID NO:34), MitP (e.g., SEQ ID NO:112 encoded by SEQ ID NO:33), MitQ (e.g., SEQ ID NO:113 encoded by SEQ ID NO:32), MitR (e.g., SEQ ID NO:114 encoded by SEQ ID NO:31), MitS (e.g., SEQ ID NO: 115 encoded by SEQ ID NO:30), MitT (e.g., SEQ ID NO:140 encoded by SEQ ID NO:29), MmcA (SEQ ID NO:116 encoded by SEQ ID NO:49), MmcB (SEQ ID NO:117 encoded by SEQ ID NO:50), MmcC (SEQ ID NO:118 encoded by SEQ ID NO:51), MmcD (SEQ ID NO:119 encoded by SEQ ID NO:52), MmcE (SEQ ID NO:120 encoded by SEQ ID NO:53), MmcF (SEQ ID NO:121 encoded by SEQ ID NO:54), MmcG (SEQ ID NO:122 encoded by SEQ ID NO:55), MmcH (SEQ ID NO:123 encoded by SEQ ID NO:56), MmcI (SEQ ID NO:124 encoded by SEQ ID NO:57), MmcJ (SEQ ID NO:125 encoded by SEQ ID NO:58), MmcK (SEQ ID NO:126 encoded by SEQ ID NO:59), MmcL (SEQ ID NO:127 encoded by SEQ ID NO:60), MmcM (SEQ ID NO:128 encoded by SEQ ID NO:61), MmcN (SEQ ID NO:129 encoded by SEQ ID NO:62), MmcO (SEQ ID NO:130 encoded by SEQ ID NO:63), MmcP (SEQ ID NO:131 encoded by SEQ ID NO:64), MmcQ (SEQ ID NO:132 encoded by SEQ ID NO:65), MmcR (SEQ ID NO:133 encoded by SEQ ID NO:66), MmcS (SEQ ID NO:134 encoded by SEQ ID NO:67), MmcT (SEQ ID NO:135 encoded by SEQ ID NO:68), MmcU (SEQ ID NO:136 encoded by SEQ ID NO:69), MmcV (SEQ ID NO:137 encoded by SEQ ID NO:70), MmcW (SEQ ID NO:138 encoded by SEQ ID NO:71), MmcX (SEQ ID NO:139 encoded by SEQ ID NO:72), MmcY (SEQ ID NO:141 encoded by SEQ ID NO:73), Mct (SEQ ID NO:117 encoded by SEQ ID NO:16), a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to at least one of the nucleic acid sequences identified hereinabove or the complement thereof.

[0016] The invention further provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a variant or a fragment thereof. Preferably, the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, and more preferably at least nine, open reading frames. More preferably, the nucleic acid molecule hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:74, or a portion thereof.

[0017] The invention also provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes sugar biosynthetic enzymes, a variant or a fragment thereof. Preferably, the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, more preferably at least nine, and even more preferably at least twelve, open reading frames. Preferably, the nucleic acid molecule of this embodiment of the invention hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:75, or a portion thereof.

[0018] The invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to a polypeptide having an amino acid sequence encoded by SEQ ID NO:76, or a fragment thereof. A preferred variant polypeptide includes a variant polypeptide or fragment thereof having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:10-12, 17 or 100-141. Thus, for example, the activity of a polypeptide having SEQ ID NO:98 can be compared to a variant of SEQ ID NO:98 having at least one amino acid substitution, insertion, or deletion relative to SEQ ID NO:98.

[0019] A variant nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:76, the complement thereof, or a fragment thereof The amino acid and/or nucleic acid similarity (or homology) of two sequences may be determined manually or using algorithms well known to the art.

[0020] The invention also provides probes and primers comprising at least a portion of the nucleic acid molecules of the invention. The probes or primers of the invention are preferably detectably labeled or have a binding site for a detectable label. Preferably, the probes or primers of the invention are at least about 7, more preferably at least about 15, contiguous nucleotides bases having at least about 80% identity, more preferably at least about 90% identity, to the isolated nucleic acid molecules of the invention. Such probes or primers are useful to detect, quantify, isolate and/or amplify DNA strands with complementary to sequences related to the mitomycin biosynthetic gene cluster, sequences related to those encoding the polyketide biosynthetic enzymes linked to the mitomycin biosynthetic gene cluster, sequences related to those encoding sugar biosynthetic enzymes linked to the mitomycin biosynthetic gene cluster, a variant or a fragment thereof.

[0021] Also provided is an expression cassette comprising a nucleic acid molecule comprising at least a portion of a mitomycin biosynthetic gene cluster, a nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes sugar biosynthetic enzymes, a variant or fragment thereof; operably linked to a promoter functional in a host cell. Host cells that have been modified genetically, i.e., recombinant host cells, include host cells comprising an expression cassette, e.g., an expression cassette of the invention, or host cells in which the genome has been genetically manipulated, e.g., by deletion of a portion of, replacement of a portion of, or by disruption of, the host chromosome, so as to reduce or eliminate the expression of a particular mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention.

[0022] One embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of a nucleic acid sequence comprising the mitomycin gene cluster, i.e., the endogenous or native genomic sequence, is disrupted or replaced, for example, by an insertion with heterologous sequences or substituted with a variant nucleic acid sequence of the invention, preferably so as to result in altered mitomycin synthesis, such as an increase in mitomycin synthesis, and/or production of a novel compound. For example, the invention includes a recombinant host cell in which the mmcW gene is disrupted, for example, by replacement with a selectable marker gene, so as to yield a recombinant host cell having an increase in mitomycin production.

[0023] Another embodiment of the invention is a recombinant host cell, the genome of which is augmented by an expression cassette, e.g., via an extrachromosomal element such as a plasmid or by stable integration of the cassette into the host chromosome. Thus, the genome of the recombinant host cell is augmented with at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell.

[0024] Alternatively, the genome of a recombinant host cell is augmented with a non-mitomycin biosynthetic gene and, optionally, at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell. For example, the recombinant host cell may be augmented with pik4 (see U.S. application Ser. No. 09/105,537, filed Jun. 26, 1998, the disclosure of which is incorporated by reference herein) and piki expressed in an amount effective to yield a novel compound(s).

[0025] Host cells usefull to prepare the recombinant host cells of the invention include cells which do not express or do not comprise nucleic acid corresponding to the nucleic acid molecules of the invention, e.g., mitomycin biosynthetic genes, as well as cells which naturally produce mitomycin.

[0026] Thus, the invention also provides isolated and purified polypeptides encoded by a nucleic acid molecule of the invention. Preferably, the polypeptide of the invention is obtained from recombinant host cells, e.g., the genome of which is augmented by a nucleic acid molecule of the invention. In addition, expression cassettes and host cells comprising antisense sequences of at least a portion of the mitomycin biosynthetic gene cluster of the invention are envisioned.

[0027] In another embodiment of the invention, the isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, e.g., a polyketide synthase, is useful in methods to prepare recombinant polyhydroxyalkanoate monomer synthases and polymers.

[0028] Thus, the present invention provides a method of preparing a polyhydroxyalkanoate synthase. The method comprises introducing expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyketide synthase, operably linked to a promoter functional in the host cell. The DNA molecule is preferably obtained from a mitomycin-producing organism, e.g., a Streptomyces spp. such as S. lavendulae. The DNA molecule encoding the polyketide synthase is then expressed in the cell. Thus, another embodiment of the invention provides a purified recombinant polyketide isolated from a host cell which expresses the synthase.

[0029] Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a host cell. The first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the host cell, e.g., an insect cell. The inactivation preferably is via a mutation in the catalytic site of the dehydrase. The second expression cassette comprises a DNA segment encoding a polyketide synthase that is preferably obtained from a mitomycin-producing organism operably linked to a promoter functional in the host cell. The expression cassettes may be on the same or separate molecules. The DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer.

[0030] The present invention also provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell. The nucleic acid molecule comprises a plurality of DNA segments. Thus, the nucleic acid molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. It is also preferred that the first DNA segment comprises a module from a mitomycin-producing organism, e.g., Streptomyces spp. The nucleic acid molecule may optionally firther comprise a third DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second nucleic acid molecule encoding a polyhydroxyalkanoate synthase may be introduced into the host cell.

[0031] Also provided is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module. Together the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase. It is preferred that no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. Also, it is preferred that no more than one module is derived from the gene cluster from Streptomyces hygroscopicus that encodes rapamycin or the gene cluster that encodes spiramycin. A preferred embodiment of the invention employs a first DNA segment comprising a module from a mitomycin-producing organism. A further preferred embodiment of the isolated DNA molecule of the invention includes a DNA segment encoding a polyhydroxyalkanoate synthase.

[0032] Further provided is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first DNA molecule and a second DNA molecule into a host cell. The first DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase. The recombinant polyhydroxyalkanoate monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first module and a second module. The first DNA molecule is operably linked to a promoter functional in a host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. It is preferred that at least one module is from a mitomycin-producing organism. The DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.

[0033] Yet another embodiment of the invention is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. That is, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase and the second DNA segment encodes a module of a polyketide synthase. A preferred embodiment of the invention employs a second DNA segment comprising a module of a polyketide synthase from a mitomycin-producing organism such as Streptomyces.

[0034] Also provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first and second module which together encode the monomer synthase. A preferred embodiment of the invention employs a module from a mitomycin-producing organism. Optionally, the expression cassette further comprises a second DNA molecule encoding a polyhydroxyalkanoate synthase.

[0035] The invention also provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase. Preferably, at least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of S. lavendulae. Also preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3′ most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.

[0036] Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. Preferably, the second DNA molecule is derived from DNA which is linked to the mitomycin gene cluster. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from DNA which is linked to a mitomycin gene cluster, e.g., the mitomycin gene cluster of S. lavendulae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.

[0037] Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the DNA which is linked to the mitomycin gene cluster of S. lavendulae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.

[0038] Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of S. lavendulae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.

[0039] Thus, the invention provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae. Preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3′ most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.

[0040] Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.

[0041] Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the mit/mmc gene cluster of S. lavendulae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.

[0042] Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.

[0043] Also provided is a method for directing the biosynthesis of specific sugar-modified polyketides by genetic manipulation of a polyketide-producing microorganism. The method comprises introducing into a polyketide-producing microorganism a DNA sequence encoding enzymes in sugar biosynthesis, e.g., a DNA sequence comprising SEQ ID NO:75, a variant or fragment thereof, so as to yield a microorganism that produces specific sugar-modified polyketides. Alternatively, an anti-sense DNA sequence of the invention may be employed. Then the sugar-modified polyketides are isolated from the microorganism. It is preferred that the DNA sequence is modified so as to result in the inactivation of at least one enzymatic activity in sugar biosynthesis or in the attachment of the sugar to a polyketide

[0044] Thus, the modules encoded by the nucleic acid segments of the invention may be employed in the methods described hereinabove to prepare polyhydroxyalkanoates of varied chain length or having various side chain substitutions.

[0045] The compounds produced by the recombinant host cells of the invention are preferably biologically active agents such as antibiotics, anti-inflammatory agents, anti-cancer agents, antibiotics, immune-enhancers, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, or cholesterol-lowering agents; or as crop protection agents (e.g., fungicides or insecticides), as well as biopolymers, e.g., in packaging or biomedical applications, or to engineer PHA monomer synthases. Methods employing these compounds, e.g., to treat a mammal, e.g., a human, bird or fish in need of such therapy, are also envisioned.

BRIEF DESCRIPTION OF THE FIGURES

[0046]FIG. 1. The biosynthetic pathway for mitomycin antibiotics.

[0047]FIG. 2. Organization of the mitomycin gene cluster. The deduced ORFs are drawn to scale, and their corresponding genes are marked in italics. The filled bars indicate the location of the mitomycin cluster. Abbreviations of the restriction enzymes: B: BamHI, S: SphI, P: PstI, E: EcoRI, X: XhoI, K: KpnI.

[0048]FIG. 3. The three SAM dependent methyltransferase conserved motifs can be found in MitM (SEQ ID NO:1), MitN (SEQ ID NO:2), and MmcR (SEQ ID NO:3). DmpM (SEQ ID NO:4; Kim et al., 1998), TcmN (SEQ ID NO:5; Shikano et al., 1998), ORF14 (SEQ ID NO:6; August et al., 1998), EryG (SEQ ID NO:7; Hardwick and Pelham, 1994) are O-methyltransferases for puromycin, tetracenomycin C, rifamycin, and erytliromycin biosynthesis, respectively. Consen=consensus sequence (SEQ ID NO:8).

[0049]FIG. 4. Sequence similarity of MitM, MitN, and MmcR with other O-methyltransferases: DmpM (Kim et al., 1998), TcmN (Shikano et al., 1998), ORF14 (August et al., 1998), EryG (Hardwick and Pelham, 1994), RdmB (Mazodier et al., 1989), DnrK (Lee and Stock, 1996), and DauK (Devereux et al. 1984)); and C-methyltransferases: SMT (Schafeijohann et al., 1993), ESMTI (Floss, 1997), SMT1 (Blattner et al., 1997), and SED6 (Guilfoile and Hutchinson, 1992)). The dendrogram was constructed with the program PILEUP (Denis and Brzezinld, 1992).

[0050]FIG. 5. MC genes and deduced enzme functions.

[0051]FIG. 6. Bacterial strains and plasmids. Strains DH5α and DH5αF′ are available from Gibco BRL (Gaithersburg, MD), ATCC 27643 and NRRL 2564 are available from the American Type Culture Collection, and strain S17-1 is described in Hidaka et al. (1995). Plasmids pNJ1, pUC119, pKC 1139, pDHS3001, pKN108, and pFD666 are described in Kuzuyama et al. (1995), Madduri et al. (1993), Boxer (1997), Kagan and Clarke (1994), Kim et al. (1998), and Coque et al. (1995), respectively.

[0052]FIG. 7. Biosynthetic pathway leading to mitomycin C.

[0053]FIG. 8. Southern hybridization and restriction-enzyme map of the mrd and rifK hybridizing regions from S. lavendulae. A) Southern hybridization with the rifK gene probe (Kim et al., 1998). Lane 1, A. mediteranei ATCC 27643 genomic DNA digested with BamHI; Lane 2, S. lavendulae NRRL 2564 genomic DNA digested with BamHI; B) Physical map showing the mitABC genes. The location of mrd and rifK hybridizing genes in cosmid pDHS7529 are indicated by solid bars. Enzymes: E, EcoRI; B, BamHI. The sequenced 3.8 kb BamHI fragment containing mitA, mitB, mitC is enlarged (wide arrows). Thin arrows below show sites of resistance gene integration for disruption experiments.

[0054]FIG. 9. Nucleotide sequence of the 3.8 kb DNA fragment containing mitABC (SEQ ID NO:9). The deduced gene products are indicated in the one-letter code under the DNA sequence (SEQ ID NO:10, MitA; SEQ ID NO: 11, MitB; SEQ ID NO:12, MitC). Possible ribosome binding sites are marked in the boxed regions. The presumed translational start site and direction of transcription for each ORF is indicated by an arrow and marked accordingly.

[0055]FIG. 10. Alignment of MitA with three other AHBA synthases. The deduced amino acid sequence comparison from AHBAS genes derived from Streptomyces lavendulae (SEQ ID NO:10). Streptomyces collinus (Z54208; SEQ ID NO:13), Actinosynnema pretiosum (I39657; SEQ ID NO:14), and Amycolatopsis mediteranei (I39657; SEQ ID NO:15) is shown with the conserved lysine in the PLP-binding motif underlined.

[0056]FIG. 11. Southern blot analysis of the mitA mutant strain. A) Construction of mitA disruption mutant and restriction map of the wild-type and mitA disruption mutant showing expected band sizes in the Southern blot. Maps are not drawn to scale. B) S. lavendulae genomic DNA from wild-type (lanes 1 and 2) and double crossover mutant (lanes 3 and 4) were digested with BamHI (lane 1 and 3) and SphI (lane 2 and lane 4), respectively. The 4.9 kb EcoRI-HindIII fragment from pDHS2001 containing tsr-disrupted mitA was used as the probe.

[0057]FIG. 12. Southern blot analysis of mitB mutant MM101. A) Construction of mitB disruption mutant and restriction map of the wild-type and mitB disruption mutant showing the expected sites in the Southern blot. B) S. lavendulae genomic DNA from wild-type (lane 1 and 3) and mitB mutant (lane 2 and 4) were digested with BamHI (lane 1 and 2) and Sacl (lane 3 and 4). DNA probe: 3.8 kb BamHI fragment insert from pDHS7601.

[0058]FIG. 13. Chemical analysis and biological activity of extracts from S. lavendulae wild-type and mutant strains. A) HPLC analysis of authentic mitomycin C standard, mitomycin C production in the wild-type S. lavendulae, mitA (AHBAS) and mitB (gtf) disruption mutants of S. lavendulae. One mg of crude extract injected, 1 μg of MC injected as standard. B) Bacillus subtilis bioassay of mitomycin C production in mitA disruption mutant strain of S. lavendulae. Filter discs: 1) 100 μg injection of wild-type—collected 12.5-13.5 minutes; 2) 100 μg injection of mitA (ahbas) disruption mutant—collected 12.5-13.5 minutes; 3) 100 μg injection of W.T. containing vector—collected 12.5-13.5 minutes; 4) One μg of mitomycin C collected from HPLC from 12.5-13.5 minutes; 5) Tris buffer negative control; 6) methanol solvent negative control.

[0059]FIG. 14. Strains and plasmids employed in Example 3. BL21 (DE3) and pET17b are available from Novagen (Madison, Wis.). pDH57006 is described in Sheldon et al. (1997).

[0060]FIG. 15. Genetic map showing the physical linkage of the mct and mrd genes within the MC biosynthetic gene cluster. The expanded box shows the line plot of the mct ORF.

[0061]FIG. 16. The nucleotide sequence of mot (SEQ ID NO:16). The deduced amino acid sequence of mct is indicated under the nucleotide sequence with the one letter designation (SEQ ID NO:17). A conserved motif characteristic of 14 TMS proteins is boxed while the invariant beta-turn motif is denoted with a dashed underline. The putative ribosome binding site is marked with a solid underline.

[0062]FIG. 17. Dot matrix alignment of the deduced amino acid sequence of mct with other actinomycete antibiotic efflux proteins. Comparable parameters were utilized in generating the alignments.

[0063]FIG. 18. Hydropathy analysis of the deduced amino acid sequence of MC-translocase. A) Hydropathy plot obtained from prediction of Kyte and Doolittle (1982). B) Schematic representation of MC-translocase protein topology. The trnnsmembrane spanning regions are marked (1-14). The initial and final amino acid positions of each transmembrane domain are indicated by small numbers. The relative position of positively (H, R, K) and negatively (D, Q) charged amino acids are indicated by a plus and minus, respectively.

[0064]FIG. 19. Creation of the mct disruption mutant. A) The chromosomal mct gene (black bar) was disrupted by inserting a neomycin resistance marker (shaded) within the gene. Following double crossover recombination, specific restriction bands are predicted to be shifted in the mct mutant genome compared to the wild-type strain. B) Southem blot analysis of the mct mutant. As expected, when probed with the 4.0 kb Bamli insert from pDHS7661, the 4.0 kb BamHI hybridization band in wild-type S. lavendulae was shifted to 5.4 kb in mct knockouts, while a 1.65 kb SacI hybridization band was shifted to 3.0 kb in size. Lane 1 and 5: wild-type genomic DNA digested with BamHI. Lane 2, 3, 4, and 6: Four double crossover colonies genomic DNA digested with BamHI. Lane 7: wild-type genomic DNA digested with SstI. Lane 8: double crossover clone 6 genomic DNA digested with SstI.

[0065]FIG. 20. MC uptake analysis of strains PJS100, PJS102, and PJS103. BL21(DE3)::pET17b vector control strain, (); strain PJS100, (▪); strain PJS102, (♦); strain PJS103, (x).

[0066]FIG. 21. Complete nucleotide sequence of the mitomycin gene cluster (SEQ ID NO:96).

[0067]FIG. 22. Complete nucleotide sequence of ORFs 1-9 (SEQ ID NO:74).

[0068]FIG. 23. Complete nucleotide sequence of ORFs 11-22 (SEQ ID NO:75).

[0069]FIG. 24. Codons for various amino acids.

[0070]FIG. 25. Exemplary amino acid substitutions.

[0071]FIG. 26. Complete nucleotide sequence of the mitomycin biosynthetic genes (SEQ ID NO:76).

[0072]FIG. 27A. Structure of selected mitomycins.

[0073]FIG. 27B. Structure of selected naturally occurring mitomycins.

[0074]FIG. 28. Synthetic scheme.

[0075]FIG. 29. ¹H spectrum of 9a-demethoxy mitomycin A (CD₃CN, 800 MHZ).

[0076]FIG. 30. ¹H-¹H COSY spectrm of 9a-demethoxy mitomycin A (CD₃CN, 800 MHZ).

[0077]FIG. 31. HMQC spectrum of 9a-demethoxy mitomycin A (CD₃CN, 800 MHZ).

[0078]FIG. 32. HMBC spectrum (2.4 ppm to 4.9 ppm) of 9a-demethoxy mitomycin A (CD₃CN, 800 MHZ).

[0079]FIG. 33. HMBC spectrum (1.6 ppm to 2.35 ppm) of 9a-demethoxy mitomycin A (CD₃CN, 800 MHZ).

[0080]FIG. 34. Low and high resolution electrospray ionization mass spectrum of 9a-demethoxy mitomycin A.

[0081]FIG. 35, UV spectrum of 9ademethoxy mitomycin A.

[0082]FIG. 36. ¹H spectrum of 9-epi-mitomycin B in CD₃CN and CD₃0D, 800 MHZ.

[0083]FIG. 37. Low and high resolution electrospray ionization mass spectrum of 9-epi-mitomycin B.

[0084]FIG. 38. Schematic of the construction of a mitM deletional mutant.

[0085]FIG. 39. MC production in wild type and various mitM and mitN mutants.

[0086]FIG. 40. Purification of MitM and MitN.

[0087]FIG. 41. Conversion of MA to MF by MitM and MitN.

[0088]FIG. 42. Late stages in mitomycin biosynthesis.

[0089]FIG. 43. Thin layer chromatogram of MM107 and MM108.

DETAILED DESCRIPTION OF THE INVENTION

[0090] Definitions

[0091] As used herein, a “Type I polyketide synthase” is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides.

[0092] As used herein, a “linker region” is an amino acid sequence present in a multifunctional protein which is less well conserved in an amino acid sequence than an amino acid sequence with catalytic activity.

[0093] As used herein, an “extender unit” catalytic or enzymatic domain is an acyl transferase in a module that catalyzes chain elongation by adding 2-4 carbon units to an acyl chain and is located carboxy-terminal to another acyl transferase. For example, an extender unit with methylmalonylCoA specificity adds acyl groups to a methylmalonylCoA molecule.

[0094] As used herein, a “polyhydroxyalkanoate” or “PHA” polymer includes, but is not limited to, linked units of related, preferably heterologous, hydroxyalkanoates such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate, 3-hydroxyheptanoate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy and 5-hydroxy counterparts.

[0095] As used herein, a “recombinant” nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.

[0096] As used herein, a “multifunctional protein” is one where two or more enzymatic activities are present on a single polypeptide.

[0097] As used herein, a “module” is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase.

[0098] As used herein, a “premature termination product” is a product which is produced by a recombinant multifunctional protein which is different than the product produced by the non-recombinant multifunctional protein. In general, the product produced by the recombinant multifunctional protein has fewer acyl groups.

[0099] As used herein, a DNA that is “derived from” a gene cluster is a DNA that has been isolated and purified in vitro from genomic DNA, or synthetically prepared on the basis of the sequence of genomic DNA.

[0100] An “antibiotic” as used herein is a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell.

[0101] An “antibiotic biosynthetic gene” is a nucleic acid, e.g., DNA, segment or sequence that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics.

[0102] An “antibiotic biosynthetic pathway” includes the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. These genes can be isolated by methods well known to the art, e.g., see U.S. Pat. No. 4,935,340.

[0103] Antibiotic-producing organisms include any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic.

[0104] The term “polyketide” as used herein refers to a large and diverse class of natural products, including but not limited to antibiotic, antifingal, anticancer, and anti-helminthic compounds.

[0105] The term “polyketide-producing microorganism” as used herein includes any microorganism that can produce a polyketide naturally or after being suitably engineered (i.e., genetically). Examples of actinomycetes that naturally produce polyketides include but are not limited to Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora erythraea, Streptomyces antibioticus, Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avermitilis, Streptomycesfradiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rimosus, Streptomyces peucetius, Streptomyces coelicolor, Streptomes glaucescens, Streptomyces roseofulvus, Streptomyces cinnamonensis, Streptomyces curacoi, and Amycolatopsis mediteranei (see Hopwood, D. A and Sherman, D. H., Annu. Rev. Genet., 24:37-66 (1990), incorporated herein by reference). Other examples of polyketide-producing microorganisms that produce polyketides naturally include various Actinomadura, Dactylosporangium and Nocardia strains.

[0106] The term “glycosylated polyketide” refers to any polyketide that contains one or more sugar residues.

[0107] The term “glycosylation-modified polyketide” refers to a polyketide having a changed glycosylation pattern or configuration relative to that particular polyketide's unmodified or native state.

[0108] The term “sugar biosynthesis genes” as used herein refers to nucleic acid sequences from organisms such as S. lavendulae that encode sugar biosynthesis enzymes and is intended to include sequences of DNA from other polyketide-producing microorganisms which are identical or analogous to those obtained from S. lavendulae.

[0109] The term “sugar biosynthesis enzymes” as used herein refers to polypeptides which are involved in the biosynthesis and/or attachment of polyketide-associated sugars and their derivatives and intermediates.

[0110] The term “polyketide-associated sugar” refers to a sugar that is known to attach to polyketides or that can be attached to polyketides by the processes described herein.

[0111] The term “sugar derivative” refers to a sugar which is naturally associated with a polyketide but which is altered relative to the unmodified or native.

[0112] The term “sugar intermediate” refers to an intermediate compound produced in a sugar biosynthesis pathway.

[0113] A “recombinant” host cell of the invention has a genome that has been manipulated in vitro so as to alter, e.g., decrease or disrupt, or, alternatively, increase, the function or activity of at least one gene, e.g., in the mitomycin biosynthetic gene cluster, of the invention.

[0114] As used herein, the “mit/mmc” or “mitomycin” gene cluster includes sequences encoding enzymes for mitosane precursor formation, mitosane ring assembly, regulation of mitomycin biosynthesis, functionalization, and resistance to mitomycin, as well as closely linked sequences encoding polyketide and sugar biosynthetic enzyes.

[0115] As used herein, the terms “isolated and/or purified” refer to in vitro isolation of a RNA, DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that is can be sequenced, replicated and/or expressed. Moreover, the nucleic acid may encode more than one polypeptide. For example, “an isolated DNA molecule encoding an AHBA synthase” is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that preferably encode a biologically active polypeptide, or a fragment or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of an AHBA synthase RNA, or hybridizes to the RNA or DNA encoding the AHBA synthase and remains stably bound under low, moderate, or stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., supra.

[0116] An antibiotic resistance-conferring gene is a nucleic acid segment that encodes an enzymatic or other activity which alone or in combination with other gene products, confers resistance to an antibiotic.

[0117] As used herein, “mitomycin” includes, but is not limited to, mitomycin A, mitomycin B, mitomycin C, porfiromycin, mitiromycin, mitomycin D, mitomycin E, mitomycin F, mitomycin G, mitomycin H, mitomycin I, mitomycin J, mitomycin L, mitomycin M, mitomycin K, albomitomycin A, isomitomycin A, KW2149, KW2149 metabolites such as M-16 and M-18, FR66979, FK973, FK317, and FR900482, as well as structural or functional equivalents thereof (“analogs”), or derivatives thereof.

[0118] As used herein, the term “derivative” means that a particular compound produced by a host cell of the invention or prepared in vitro using polypeptides encoded by the nucleic acid molecules of the invention, is modified so that it comprises other moieties, e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters small chemicals, metals, salts, synthetic polymers, e.g., polylactide and polyglycolide, surfactants and glycosaminoglycans, which are covalently or non-covalently attached or linked to the compound.

[0119] It will be appreciated by those skilled in the art that each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.

[0120] The term “sequence homology” or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred. When using oligonucleotides as probes, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%).

[0121] Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, 1972, volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.

[0122] The following terms are used to describe the sequence relationships between two or more polynucleotides: “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”, and “substantial identity”. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.

[0123] A “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appi. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 4& 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

[0124] The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0125] As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 80 percent sequence identity, preferably at least about 90 percent sequence identity, more preferably at least about 95 percent sequence identity, and most preferably at least about 99 percent sequence identity.

[0126] In accordance with the present invention, there is provided a purified and isolated nucleic acid molecule which encodes the entire pathway for the biosynthesis of mitomycin, as well as polyketide biosynthetic and sugar biosynthetic genes that are linked to the mitomycin biosynthetic genes. Desirably, the nucleic acid molecule is a DNA isolated from Streptomyces spp. The present invention fuirther includes isolated and purified DNA sequences which hybridize under standard or stringent conditions to the the nucleic acid molecules of the invention. It should be understood to those skilled in the art that the invention also encompasses the purified and isolated polypeptides which may be encoded by the sequences of the nucleic acid molecules of this invention.

[0127] The invention described herein can be used for the production of mitomycin, analogs or derivatives thereof, or novel compounds. Commercial chemical syntheses of mitomycin are not feasible. The gene cluster described herein contains all the genes required for the production of the mitosane group of antibiotics, compounds which are clinically prescribed antitumor compounds employed in the treatment of a wide variety of cancers including non-small cell lung cancer, metastatic breast cancer, esophageal, gastric, pancreatic, and anal canal carcinomas. Thus, the isolation and characterization of this gene cluster allows for the selective production of mitomycin antibiotics, the overproduction or under production of particular compounds, e.g., overproduction of certain mitomycin antibiotics, and the production of novel compounds, e.g., mitomycin-derived compounds as well as the production of novel non-mitomycin related compounds. For example, combinational biosynthetic-based modification of mitomycin antibiotics may be accomplished by selective activation or disruption of specific genes within the cluster or incorporation of the genes into biased biosynthetic libraries which are assayed for a wide range of biological activities, to derive greater chemical diversity in the mitomycins. A further example includes the introduction of a mitomycin biosynthetic gene(s) into a particular host cell so as to result in the production of a novel non-mitomycin related compound due to the activity of the mitomycin biosynthetic gene(s) on other metabolites, intermediates or components of the host cells. The in vitro expression of polypeptides from this gene cluster also provides an enzymatic route to the production of known mitomycin compounds that are produced in low quantities, or conversion of currently available mitomycins to other known or novel mitomycins, e.g., the bioconversion of mitomycin C to porfiromycin.

[0128] The mitomycin resistance genes may also be used to provide higher mitomycin resistance to cancer patients undergoing treatment and for clonal selection purposes (e.g., using mrd). For example, the resistance gene(s) may be inserted into human bone marrow cell lines to confer higher resistance to non-cancerous cells, thus allowing higher doses of mitomycins to be administered to cancer patients. Moreover, because mitomycin acts directly upon DNA itself, its toxicity is extremely broad, and therefore the resistance genes could be used for efficient selection in prokaryotes, fungi, plants, mammalian cell culture, and insect cell culture. Further, the regulatory resistance and transport genes may be used to create higher producing strains capable of synthesizing more mitomycin than can currently be obtained through traditional fermentation strategies.

[0129] In addition, the invention described herein can be used for the production of novel compounds which include a diverse range of biodegradable PHA polymers through genetic redesign of DNA such as that found in Streptomyces spp. Different PHA synthases can thenbe tested for their ability to polymerize the monomers produced by the recombinant PHA synthase into a biodegradable polymer. PHA synthases can be tested for their specificity with respect to different monomer substrates by methods well known to the art.

[0130] The potential uses and applications of PHAs produced by PHA monomer synthases and PHA synthases include both medical and industrial applications. Medical applications of PHAs include surgical pins, sutures, staples, swabs, wound dressings, blood vessel replacements, bone replacements and plates, stimulation of bone growth by piezoelectric properties, and biodegradable carrier for long-term dosage of pharmaceuticals. Industrial applications of PHAs include disposable items such as baby diapers, packaging containers, bottles, wrappings, bags, and films, and biodegradable carriers for long-teni dosage of herbicides, fungicides, insecticides, or fertilizers.

[0131] In animals, the biosynthesis of fatty acids de novo from malonyl-CoA is catalyzed by FAS. For example, the rat FAS is a homodimer with a subunit structure consisting of 2505 amino acid residues having a molecular weight of 272,340 Da. Each subunit consists of seven catalytic activities in separate physical domains (Amy et al., Proc Natl. Acad. Sci. USA, 3114 (1989)). The physical location of six of the catalytic activities, ketoacyl synthase (KS), malonyl/acetyltransferase (M/AT), enoyl reductase (ER), ketoreductase (KR), acyl carrier protein (ACP), and thioesterase (TE), has been established by (1) the identification of the various active site residues within the overall amino acid sequence by isolation of catalytically active fragments from limited proteolytic digests of the whole FAS, (2) the identification of regions within the FAS that exhibit sequence similarity with various monofunctional proteins, (3) expression of DNA encoding an amino acid sequence with catalytic activity to produce recombinant proteins, and (4) the identification of DNA that does not encode catalytic activity, i.e., DNA encoding a linker region. (Smith et al., Proc. Natl Acad Sci. USA, 73, 1184 (1976); Tsukamoto et al., J. Biol. Chem., 2M, 16225 (1988); Rangan et al., J. Biol Cem, 266, 19180 (1991)).

[0132] The seventh catalytic activity, dehydrase (DH), was identified as physically residing between AT and ER by an amino acid comparison of FAS with the amino acid sequences encoded by the three open reading frames of the eryA polyketide synthase (PKS) gene cluster of Saccharopolyspora erythraea. The three polypeptides that comprise this PKS are constructed from “modules” which resemble animal FAS, both in terms of their amino acid sequence and in the ordering of the constituent domains (Donadio et al., Gene, 111, 51 (1992); Benh et al., Fur. J. Biochem., 204, 39 (1992)).

[0133] One embodiment of the invention employs a FAS in which the DH is inactivated (FAS DH-). The FAS DH-employed in this embodiment of the invention is preferably a eukaryotic FAS DH- and, more preferably, a mammalian FAS DH-. The most preferred embodiment of the invention is a FAS where the active site in the DH has been inactivated by mutation. For example, Joshi et al. (J. Biol. Chem., 268, 22508 (1993)) changed the His⁸⁷⁸ residue in the rat FAS to an alanine residue by site-directed mutagenesis. In vitro studies showed that a FAS with this change (ratFAS206) produced 3-hydroxybutyrylCoA as a premature termination product from acetyl-CoA, malonyl-CoA and NADPH.

[0134] A FAS DH-effectively replaces the β-ketothiolase and acetoacetyl-CoA reductase activities of the natural pathway by producing D(−)-3-hydroxybutyrate as a premature termination product, rather than the usual 16-carbon product, palmitic acid. This premature termination product can then be incorporated into PHB by a PHB synthase.

[0135] Another embodiment of the invention employs a recombinant Streptomyces spp. PKS to produce a variety of β-hydroxyCoA esters that can serve as monomers for a PHA synthase. One example of a DNA encoding a Type I PKS is the eryA gene cluster, which governs the synthesis of erytliromycin aglycone deoxyeiythronolide B (DEB). The gene cluster encodes six repeated units, termed modules or synthase units (SUs). Each module or SU, which comprises a series of putative FAS-like activities, is responsible for one of the six elongation cycles required for DEB formation. Thus, the processive synthesis of asymmetric acyl chains found in complex polyketides is accomplished through the use of a programmed protein template, where the nature of the chemical reactions occurring at each point is determined by the specificities in each SU.

[0136] Two other Type I PKS are encoded by the tyl (tylosin) and met (methymycin) gene clusters (see U.S. application Ser. No. 09/108,537, the disclosure of which is incorporated by reference herein). The macrolide multifunctional synthases encoded by tyl and met provide a greater degree of metabolic diversity than that found in the eryA gene cluster. The PKSs encoded by the eryA gene cluster only catalyze chain elongation with methylmalonylCoA, as opposed to tyl and met PKSs, which catalyze chain elongation with malonylCoA, methylmalonylCoA and ethylmalonylCoA. Specifically, the tyl PKS includes two malonylCoA extender units and one ethylmalonylCoA extender unit, and the met PKS includes one malonylCoA extender unit.

[0137] In order to manipulate the catalytic specificities within each module, DNA encoding a catalytic activity must remain undisturbed. To identify the amino acid sequences between the amino acid sequences with catalytic activity, the “linker regions,” amino acid sequences of related modules, preferably those encoded by more than one gene cluster, are compared. Linker regions are amino acid sequences which are less well conserved than amino acid sequences with catalytic activity. Witkowski et al., Eur. J. Biochem., 198, 571 (1991).

[0138] In an alternative embodiment of the invention, to provide a DNA encoding a Type I PKS module with a TE and lacking a functional DH, a DNA encoding a module F, containing KS, MT, KR, ACP, and TE catalytic activities, is introduced at the 3′ end of a DNA encoding a first module. Module F introduces the final (R)-3-hydroxyl acyl group at the final step of PHA monomer synthesis, as a result of the presence of a TE domain. DNA encoding a module F is not present in the eryA PKS gene cluster (Donadio et al., supra, 1991).

[0139] A DNA encoding a recombinant monomer synthase is inserted into an expression vector. The expression vector employed varies depending on the host cell to be transformed with the expression vector. That is, vectors are employed with transcription, translation and/or post-translational signals, such as targeting signals, necessary for efficient expression of the genes in various host cells into which the vectors are introduced. Such vectors are constructed and transformed into host cells by methods well known in the art. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1989). Preferred host cells for the vectors of the invention include insect, bacterial, and plant cells. Preferred insect cells include Spodoptera frugiperda cells such as Sf21, and Trichoplusia ni cells. Preferred bacterial cells include Escherichia coli, Streptomyces and Pseudomonas. Preferred plant cells include monocot and dicot cells, such as maize, rice, wheat, tobacco, legumes, carrot, squash, canola, soybean, potato, and the like.

[0140] Moreover, the appropriate subcellular compartment in which to locate the enzyme in eukaryotic cells must be considered when constructing eukaryotic expression vectors. Two factors are important: the site of production of the acetyl-CoA substrate, and the available space for storage of the PHA polymer. To direct the enzyme to a particular subcellular location, targeting sequences may be added to the sequences encoding the recombinant molecules.

[0141] The baculovirus system is particularly amenable to the introduction of DNA encoding a recombinant FAS or a PKS monomer synthase because an increasing variety of transfer plasmids are becoming available which can accommodate a large insert, and the virus can be propagated to high titers. Moreover, insect cells are adapted readily to suspension culture, facilitating relatively large-scale recombinant protein production. Further, recombinant proteins tend to be produced exclusively as soluble proteins in insect cells, thus, obviating the need for refolding, a task that might be particularly daunting in the case of a large multifunctional protein. The Sf21/baculovirus system has routinely expressed milligram quantities of catalytically active recombinant fatty acid synthase. Finally, the baculoviruslinsect cell system provides the ability to construct and analyze different synthase proteins for the ability to polymerize monomers into unique biodegradable polymers.

[0142] A further embodiment of the invention is the introduction of at least one DNA encoding a PHA synthase and a DNA encoding a PHA monomer synthase into a host cell. Such synthases include, but are not limited to, A. eutrophus 3-hydroxy, 4-hydroxy, and 5-hydroxy alkanoate synthases, Rhodococcus ruber C₃-C₅ hydroxyalkanoate synthases, Pseudomonas oleororans C₆-C₁₄ hydroxyalkanoate synthases, P. putida C ₆-C₁₄ hydroxyalkanoate synthases, P. aeruginosa C₅-C₁₀ hydroxyalkanoate synthases, P. resinovorans C₄-C₁₀ hydroxyalkanoate synthases, Rhodospirillum rubrum C₄-C₇ hydroxyalkanoate syntheses, R. gelatinorus C₄-C₇ , Thiocapsa pfennigii C₄-C₈ hydroxyalkanoate synthases, and Bacillus megaterium C₄-C₅ hydroxyalkanoate synthases.

[0143] The introduction of DNA(s) encoding more than one PHA synthase may be necessary to produce a particular PHA polymer due to the specificities exhibited by different PHA synthases. As multifunctional proteins are altered to produce unusual monomeric structures, synthase specificity may be problematic for particular substrates. Although the A. eutrophus PHB synthase utilizes only C4 and C5 compounds as substrates, it appears to be a good prototype synthase for initial studies since it is known to be capable of producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (Kunioka et al., Macromolecules, 22, 694 (1989)) as well as copolymers of 3-hydroxyvalerate, 3-hydroxybutyrate, and 5-hydroxyvalerate (Doi et al., Macromolecules, 19, 2860 (1986)). Other synthases, especially those of Pseudomonas aeruginosa (Timm et al., Eur. J. Biochem., 209, 15 (1992)) and Rhodococcus ruber (Pieper et al., FEMS Microbiol. Lett., 96, 73 (1992)), can also be employed in the practice of the invention. Synthase specificity may be alterable through molecular biological methods.

[0144] In yet another embodiment of the invention, a DNA encoding a FAS and a PRA synthase can be introduced into a single expression vector, obviating the need to introduce the genes into a host cell individually.

[0145] A further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS module F. Module F will carry out the final chain extension to include two additional carbons and the reduction of the β-keto group, which results in a (R)-3-hydroxy acyl CoA moiety.

[0146] To produce this recombinant protein, DNA encoding the FAS TE is replaced with a DNA encoding a linker region which is normally found in the ACP-KS interdomain region of bimodular ORFs. DNA encoding a module F is then inserted 3′ to the DNA encoding the linker region. Different linker regions, such as those described below which vary in length and amino acid composition, can be tested to determine which linker most efficiently mediates or allows the required transfer of the nascent saturated fatty acid intermediate to module F for the final chain elongation and keto reduction steps. The resulting DNA encoding the protein can then be tested for expression of long-chain β-hydroxy fatty acids in insect cells, such as Sf21 cells, or Streptomyces, or Pseudomonas. The expected 3-hydroxy C-18 fatty acid can serve as a potential substrate for PHA synthases which are able to accept long-chain alkyl groups. A preferred embodiment of the invention is a FAS that has a chain length specificity between 4-22 carbons.

[0147] Examples of linker regions that can be employed in this embodiment of the invention include, but are not limited to, the ACP-KS linker regions encoded by the tyl ORFI (ACP₁-KS₂; ACP₂-KS₃), and ORF3 (ACP₅-KS₆), and eryA ORFI (ACP₁-KS₁; ACP₂-KS₂), ORF2 (ACP₃-KS₄) and ORF3 (ACP₅-KS₆).

[0148] This approach can also be used to produce shorter chain fatty acid groups by limiting the ability of the FAS unit to generate long-chain fatty acids. Mutagenesis of DNA encoding various FAS catalytic activities, starting with the KS, may result in the synthesis of short-chain (R)-3-hydroxy fatty acids.

[0149] The PHA polymers are then recovered from the biomass. Large-scale solvent extraction can be used, but is expensive. An alternative method involving heat shock with subsequent enzymatic and detergent digestive processes is also available (Byron, Trends Biotechnical, 5, 246(1987); Holmes, In: Developments in Crystalline Polymers, D. C. Bassett (ed.), pp. 1-65 (1988)). PHB and other PHAs are readily extracted from microorganisms by chlorinated hydrocarbons. Refluxing with chloroform has been extensively used; the resulting solution is filtered to remove debris and concentrated, and the polymer is precipitated with methanol or ethanol, leaving low-molecular-weight lipids in solution. Longer side-chain PHAs show a less restricted solubility than PHB and are, for example, soluble in acetone. Other strategies adopted include the use of ethylene carbonate and propylene carbonate as disclosed by Lafferty et al. (Chem, Rundschau, 30, 14 (1977)) to extract PHB from biomass. Scandola et al. (Int. J. Biol. Microbiol., 10, 373 (1988)) reported that 1 M HCl-chloroform extraction of Rhizobium meliloti yielded PHB of M_(W)=6×10⁴ compared with 1.4 ×10⁶ when acetone was used.

[0150] Methods are well known in the art for the determination of the PHB or PHA content of microorganisms, the composition of PHAs, and the distribution of the monomer units in the polymer. Gas chromatography and high-pressure liquid chromatography are widely used for quantitative PHB analysis. See Anderson et al., Microbiol. Rev, 54, 450 (1990) for a review of such methods. NMR techniques can also be used to determine polymer composition, and the distribution of monomer units.

[0151] Variants of the Nucleic Acid Molecules of the Invention

[0152] The present invention contemplates nucleic acid sequences which hybridize under low, medium or high stringency hybridization conditions to the exemplified nucleic acid sequences set forth herein. Hybridization conditions are well known in the art. Thus, nucleic acid sequences encoding variant polypeptides, i.e., those having at least one amino acid substitution insertion, addition or deletion, or nucleic acid sequences having conservative (e.g., silent) nucleotide substitutions (see FIGS. 24-25), are within the scope of the invention. Preferably, variant polypeptides encoded by the nucleic acid sequences of the invention are biologically active. The present invention also contemplates naturally occurring allelic variations and mutations of the nucleic acid sequences described herein.

[0153] As is well known in the art, because of the degeneracy of the genetic code, there are numerous other DNA and RNA molecules that can code for the same polypeptides as those encoded by the exemplified biosynthetic genes and fragments thereof The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode the polypeptides of, for example, portions of SEQ ID NO:96. Having identified the amino acid residue sequence encoded by a mitomycin, sugar or polyketide biosynthetic gene, and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention.

[0154] The 20 common amino acids and their representative abbreviations, symbols and codons are well known in the art (see, for example, Molecular Biology of the Cell, Second Edition, B. Alberts et al., Garland Publishing Inc., New York and London, 1989). As is also well known in the art, codons constitute triplet sequences of nucleotides in mRNA molecules and as such, are characterized by the base uracil (U) in place of base thymidine (T) which is present in DNA molecules. A simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure of the encoded polypeptide. By way of example, it canbe seen from SEQ ID NO:16 that a TCA codon for serine exists at nucleotide positions 146-148. However, serine can be encoded by a TCT codon, and a TCC codon. Substitution of the latter codons for serine with the TCA codon for serine or vice versa, does not substantially alter the DNA sequence of SEQ ID NO:16 and results in production of the same polypeptide. In a similar manner, substitutions of the recited codons with other equivalent codons can be made in a like manner without departing from the scope of the present invention.

[0155] A nucleic acid molecule, segment or sequence of the present invention can also be an RNA molecule, segment or sequence. An RNA molecule contemplated by the present invention corresponds to, is complementary to or hybridizes under low, medium or high stringency conditions to, any of the DNA sequences set forth herein. Exemplary and preferred RNA molecules are mRNA molecules that comprise at least one mitomycin, sugar or polyketide biosynthetic gene of this invention.

[0156] Mutations can be made to the native nucleic acid sequences of the invention and such mutants used in place of the native sequence, so long as the mutants are able to function with other sequences to collectively catalyze the synthesis of an identifiable sugar, polyketide or mitomycin. Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. Proc. Natl. Acad, Sci, USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.) Alternatively, the mutations can be effected using a mismatched primer (generally 10-30 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. Zoller and Smith, Methods Enzymol., (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409. PCR mutagenesis will also find use for effecting the desired mutations.

[0157] Random mutagenesis of the nucleotide sequence can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.

[0158] Large populations of random enzyme variants can be constructed in vivo using “recombination-enhanced mutagenesis.” This method employs two or more pools of, for example, 10⁶ mutants each of the wild-type encoding nucleotide sequence that are generated using any convenient mutagenesis technique and then inserted into cloning vectors.

[0159] Chimeric Expression Cassettes, Vectors and Host Cells of the Invention

[0160] As used herein, “chimeric” means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the “native” or wild type of the species. The recombinant DNA sequence or segment, used for transformation herein, may be circular or linear, double-stranded or single-stranded. Generally, the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA present in the resultant transformed (recombinant) host cell. Aside from DNA sequences that serve as transcription units for the nucleic acid molecules of the invention or portions thereof, a portion of the DNA may be untranscribed, serving a regulatory or a structural function. For example, the preselected DNA may itself comprise a promoter that is active in a particular host cell.

[0161] Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the MRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

[0162] “Control sequences” is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotic cells, for example, include a promoter, and optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Other regulatory sequences may also be desirable which allow for regulation of expression of the genes relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Othertypes of regulatory elements may also be present in the vector, for example, enhancer sequences.

[0163] “Operably linked” is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

[0164] The DNA to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroa, dapa and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Pat. No. 5,848,956).

[0165] Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0166] Prokaryotic expression systems are preferred, and in particular, systems compatible with Streptomyces spp. are of particular interest. Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites. Particularly useful promoters include control sequences derived from the gene clusters of the invention. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the expression cassettes encoding desosamine. Preferred promoters are Streptomyces promoters, including but not limited to the ermE*, pita and tipA promoters. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the β-lactamase (bla) promoter system, bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No 4,551,433), which do not occur in nature, also fimction in bacterial host cells.

[0167] The various nucleic acid molecules of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter. The nucleic acid molecules can include flanking restriction sites to allow for the easy deletion and insertion of other sequences. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques, such as site-directed mutagenesis and PCR.

[0168] For sequences generated by random mutagenesis, the choice of vector depends on the pool of mutant sequences, i.e., donor or recipient, with which they are to be employed. Furthermore, the choice of vector determines the host cell to be employed in subsequent steps of the claimed method. Any transducible cloning vector can be used as a cloning vector for the donor pool of mutants. It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell. Phagemids and cosmids, for example, are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in M13 phage and λ phage, respectively. Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles. Cosmids which will find use in this method generally include λ phage-based vectors into which cos sites have been inserted. Recipient pool cloning vectors can be any suitable plasmid. The cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction.

[0169] Thus, for example, the cloning vector employed may be an E. coli/Streptomyces shuttle vector (see, for example, U.S. Pat. Nos. 4,416,994, 4,343,906,4,477,571, 4,362,816, and 4,340,674), a cosmid, a plasmid, an artificial bacterial chromosome (see, e.g., Zhang and Wing, Plant Mol. Biol, 35, 115 (1997); Schalkwyk et al., Curr Op. Biotech., 6, 37 91995); and Monaco and Lavin, Trends in Biotech., 12, 280 (1994), or a phagemid, and the host cell may be a bacterial cell such as E. coli, Penicillium patulum, and Streptomyces spp. such as S. lividans, S. venezuelae, or S. lavendulae, or a eukaryotic cell such as fungi, yeast or a plant cell, e.g., monocot and dicot cells, preferably cells that are regenerable.

[0170] The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein. For example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989), provides suitable methods of construction.

[0171] The recombinant DNA can be readily introduced into the host cells by any procedure useful for the introduction into a particular cell, e.g., calcium phosphate precipitation, protoplast fusion, conjugation, lipofection, electroporation, and the like.

[0172] As used herein, the term “cell line” or “host cell” is intended to refer to well-characterized homogenous, biologically pure populations of cells. These cells may be eukaryotic cells that are neoplastic or which have been “immortalized” in vitro by methods known in the art, as well as primary cells, or prokaryotic cells. In particular, the cell line or host cell may be of mammalian, plant, insect, yeast, fungal or bacterial origin.

[0173] “Transfected” or “transformed” is used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one DNA sequence, which DNA is also referred to in the art of genetic engineering as “heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “genetically engineered,” “non-native,” or “foreign DNA,” wherein said DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering. The transfected DNA may be maintained as an extrachromosomal element or as an element which is stably integrated into the host chromosome.

[0174] Moreover, recombinant polypeptides having a particular activity may be prepared via “gene-shuffling”. See, for example, Crameri et al., Nature, 391, 288(1998); Patten et al., Curr. Op. Biotech., 8, 724(1997), U.S. Pat. Nos. 5,837,458, 5,834,252, 5,830,727, 5,811,238, 5,605,793).

[0175] For phagemids, upon infection of the host cell which contains a phagemid, single-stranded phagemid DNA is produced, packaged and extruded from the cell in the form of a transducing phage in a manner similar to other phage vectors. Thus, clonal amplification of mutant encoding nucleotide sequences carried by phagemids is accomplished by propagating the phagemids in a suitable host cell.

[0176] Following clonal amplification, the cloned donor pool of mutants is infected with a helper phage to obtain a mixture of phage particles containing either the helper phage genome or phagemids mutant alleles of the wild-type encoding nucleotide sequence.

[0177] Infection, or transfection, of host cells with helper phage is generally accomplished by methods well known in the art (see., e.g., Sambrook et al., supra; and Russell et al. (1986) Gene 4:333-338).

[0178] The helper phage may be any phage which can be used in combination with the cloning phage to produce an infective transducing phage. For example, if the cloning vector is a cosmid, the helper phage will necessarily be a λ phage. Preferably, the cloning vector is a phagemid and the helper phage is a filamentous phage, and preferably phage M13.

[0179] If desired after infecting the phagemid with helper phage and obtaining a mixture of phage particles, the transducing phage can be separated from helper phage based on size difference (Barnes et al. (1983) Methods Enzymol. 1(1:98-122), or other similarly effective technique.

[0180] The entire spectrum of cloned donor mutations can now be transduced into clonally amplified recipient cells into which has been transduced or transformed a pool of mutant encoding nucleotide sequences. Recipient cells which may be employed in the method disclosed and claimed herein may be, for example, E. coli, or other bacterial expression systems which are not recombination deficient. A recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as rec mutants of E. coli (see, Clark et al. (1965) Proc. Natl. Acad. Sci. USA 53:451-459).

[0181] These transductants can now be selected for the desired expressed protein property or characteristic and, if necessary or desirable, amplified. Optionally, if the phagemids into which each pool of mutants is cloned are constructed to express different genetic markers, as described above, transductants may be selected by way of their expression of both donor and recipient plasmid markers

[0182] The recombinants generated by the above-described methods can then be subjected to selection or screening by any appropriate method, for example, enzymatic or other biological activity.

[0183] The above cycle of amplification, infection, transduction, and recombination may be repeated any number of times using additional donor pools cloned on phagemids. As above, the phagemids into which each pool of mutants is cloned may be constructed to express a different marker gene. Each cycle could increase the number of distinct mutants by up to a factor of 10⁶. Thus, if the probability of occurrence of an inter-allelic recombination event in any individual cell is f (a parameter that is actually a function of the distance between the recombining mutations), the transduced culture from two pools of 10⁶ allelic mutants will express up to 10¹² distinct mutants in a population of 10¹²/fcells.

[0184] Preparation, Isolation and Modification of the Polypeptides of the Invention

[0185] The present isolated, purified polypeptides, variants or fragments thereof, can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant DNA approaches (see above). The solid phase peptide synthetic method is an established and widely used method, which is described in the following references: Stewart et al., Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc., 85 2149 (1963); Meienhofer in “Hormonal Proteins and Peptides,” ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; Bavaay and Merrifield, “The Peptides,” eds. E. Gross and F. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285; and Clark-Lewis et al., Meth. Enzymol, 287, 233 (1997). These polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.

[0186] In particular, fusion polypeptides are prepared which comprise an amino acid sequence useful in purification, e.g., a His tag is useful to purify fusion polypeptides on nickel columns. Once isolated and characterized, derivatives, e.g., chemically derived derivatives, of a given polypeptide can be readily prepared. For example, amides of the polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. A preferred method for amide formation at the C-terminal carboxyl group is to cleave the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.

[0187] Salts of carboxyl groups of a polypeptide or polypeptide variant of the invention may be prepared in the usual manner by contacting the polypeptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.

[0188] N-acyl derivatives of an amino group of the polypeptide or polypeptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.

[0189] One or more of the residues of the polypeptide can be altered, so long as the polypeptide variant is biologically active. For example, it is preferred that the variant has at least about 1% of the biological activity of the corresponding non-variant polypeptide, e.g. Conservative amino acid substitutions are preferred—that is, for example, aspartic-glutamic as acidic amino acids; lysinelarginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/thereonine as hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfurontaining side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide variant.

[0190] Conservative substitutions are shown in FIG. 25 under the heading of exemplary substitutions. More preferred substitutions are under the heading of preferred substitutions. After the substitutions are introduced, the variants are screened for biological activity.

[0191] Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

[0192] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0193] (2) neutral hydrophilic: cys, ser, thr;

[0194] (3) acidic: asp, glu;

[0195] (4) basic: asn, gln, his, lys, arg;

[0196] (5) residues that influence chain orientation: gly, pro; and

[0197] (6) aromatic; trp, tyr, phe.

[0198] The invention also envisions polypeptide variants with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another.

[0199] Acid addition salts of the polypeptide or variant polypeptide or of amino residues of the polypeptide or variant polypeptide may be prepared by contacting the polypeptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art.

[0200] Antibodies of the Invention

[0201] The antibodies of the invention are prepared by using standard techniques. To prepare polyclonal antibodies or “antisera,” an animal is inoculated with an antigen that is an isolated and purified polypeptide of the invention, and immunoglobulins are recovered from a fluid, such as blood serum, that contains the immunoglobulins, after the animal has had an immune response. For inoculation, the antigen is preferably bound to a carrier peptide and emulsified using a biologically suitable emulsifying agent, such as Freund's incomplete adjuvant. A variety of mammalian or avian host organisms may be used to prepare polyclonal antibodies

[0202] Following immunization, Ig is purified from the immunized bird or mammal, e.g., goat, rabbit, mouse, rat, or donkey and the like. For certain applications, it is preferable to obtain a composition in which the antibodies are essentially free of antibodies that do not react with the immunogen. This composition is composed virtually entirely of the high titer, monospecific, purified polyclonal antibodies to the antigen. Antibodies can be purified by affinity chromatography. Purification of antibodies by affinity chromatography is generally known to those skilled in the art (see, for example, U.S. Pat. No. 4,533,630). Briefly, the purified antibody is contacted with the purified polypeptide, or a peptide thereof, bound to a solid support for a sufficient time and under appropriate conditions for the antibody to bind to the polypeptide or peptide. Such time and conditions are readily determinable by those skilled in the art. The unbound, unreacted antibody is then removed, such as by washing. The bound antibody is then recovered from the column by eluting the antibodies, so as to yield purified, monospecific polyclonal antibodies.

[0203] Monoclonal antibodies can be also prepared, using known hybridoma cell culture techniques. In general, this method involves preparing an antibody-producing fused cell line, e.g., of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species, such as a murine species, from which the myeloma cell line used was derived or is compatible. Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and sensitive and relatively “pure” immunochemically. Immunologically active fragments of the present antibodies are also within the scope of the present invention, e.g., the F(ab) fragment, scFv antibodies, as are partially humanized monoclonal antibodies.

[0204] Thus, it will be understood by those skilled in the art that the hybridomas herein referred to may be subject to genetic mutation or other changes while still retaining the ability to produce monoclonal antibody of the same desired specificity. The present invention encompasses mutants, other derivatives and descendants of the hybridomas.

[0205] It will be further understood by those skilled in the art that a monoclonal antibody may be subjected to the techniques of recombinant DNA technology to produce other derivative antibodies, humanized or chimeric molecules or antibody fragments which retain the specificity of the original monoclonal antibody. Such techniques may involve combining DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of the monoclonal antibody with DNA coding the constant regions, or constant regions plus framework regions, of a different immunoglobulin, for example, to convert a mouse-derived monoclonal antibody into one having largely human immunoglobulin characteristics (see EP 184187A, 2188638A, herein incorporated by reference).

[0206] The antibodies of the invention are useful for detecting or determining the presence or amount of a polypeptide of the invention in a sample. The antibodies are contacted with the sample for a period of time and under conditions sufficient for antibodies to bind to the polypeptide so as to form a binary complex between at least a portion of said antibodies and said polypeptide. Such times, conditions and reaction media can be readily determined by persons skilled in the art.

[0207] For example, the cells are lysed to yield an extract which comprises cellular proteins. Alternatively, intact cells are permneabilized in a manner which permits macromolecules, i.e., antibodies, to enter the cell. The antibodies of the invention are then incubated with the protein extract, e.g., in a Western blot, or permeabilized cells, e.g., prior to flow cytometry, so as to form a complex. The presence or amount of the complex is then determined or detected.

[0208] The antibodies of the invention may also be coupled to an insoluble or soluble substrate. Soluble substrates include proteins such as bovine serum albumin. Preferably, the antibodies are bound to an insoluble substrate, i.e., a solid support. The antibodies are bound to the support in an amount and manner that allows the antibodies to bind the polypeptide (ligand). The amount of the antibodies used relative to a given substrate depends upon the particular antibody being used, the particular substrate, and the binding efficiency of the antibody to the ligand. The antibodies may be bound to the substrate in any suitable manner. Covalent, noncovalent, or ionic binding may be used. Covalent bonding can be accomplished by attaching the antibodies to reactive groups on the substrate directly or through a linking moiety.

[0209] The solid support may be any insoluble material to which the antibodies can be bound and which may be conveniently used in an assay of the invention. Such solid supports include permeable and semipermeable membranes, glass beads, plastic beads, latex beads, plastic microtiter wells or tubes, agarose or dextran particles, sepharose, and diatomaceous earth. Alternatively, the antibodies may be bound to any porous or liquid permeable material, such as a fibrous (paper, felt etc.) strip or sheet, or a screen or net. A binder may be used as long as it does not interfere with the ability of the antibodies to bind the ligands.

[0210] The invention will be further described by the following examples.

EXAMPLE 1 Molecular Characterization and Analysis of the mit/mmc Biosynthetic Gene Cluster

[0211] Materials and Methods

[0212] Bacterial Strains and Cloning Vectors

[0213]S. lavendulae NRRL 2564 was used as the source strain for cosmid library construction and the creation of gene disruption mutants. E. coli DH5α was used as the host strain for constructing the library and subsequent DNA manipulation. E. coli strain S17-1 (Mazodier et al., 1989) served as the conjugative host for introducing foreign DNA into S. lavendulae. The cosmid library was constructed with the E. coli/Streptomyces shuttle vector pNJ1 (Tuan et al., 1990), and pUC119 was routinely used as a vector for subcloning and sequencing. The conjugative E. coli/Streptomyces shuttle vector pKC1139 (Bierman et al., 1992) was used for gene disruption in S. lavendulae.

[0214] DNA Manipulation

[0215] Standard in vitro techniques were used for DNA manipulation (Sambrook et al., 1989). S. lavendulae genomic DNA was harvested by standard procedures (Hopwood et al., 1985).

[0216] A library of size-fractionated genomic DNA in pNJ1 (Tuan et al., 1990) was screened with the rifamycin AHBA synthase (rifK) gene probe from Amycolatopsis mediterranei (Kim et al., 1998). Through subsequent cosmid walking, a contiguous 120 kb region of S. lavendulae chromosomal DNA containing the putative mitomycin biosynthetic genes was mapped. M13 forward and reverse primers were used for sequencing (Gibco BRL, Gaithersburg, Md.). To accomplish this, individual fragments of less than 5 kb were subcloned into pUC119 and serial deletion subclones were generated using the exonuclease III Erase-a Base System (Promega, Madison, Wis.).

[0217] DNA Sequencing and Analysis.

[0218] Automatic DNA sequencing was done with the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Warrington, U.K.), and analyzed on an Applied Biosystems mode 377 DNA Sequencer at the University of Minnesota Advanced Genetic Analysis Center. Both DNA strands were sequenced redundantly a minimum of three times. Sequence compilation was performed with MacVector (Oxford Molecular Group, Mountain View, Calif.) and GeneWorks (Oxford Molecular Group) software, and sequence homology analysis was accomplished with Blast (Altschul et al., 1990) and GCG programs (Devereux et al., 1984).

[0219] Disruption Mutants Constrcion

[0220] A 1.4 kb ApaL1-HindIII fragment from pFD666 (Denis and Brzezinski, 1998) containing the aphil gene for kanamycin resistance was routinely used as the selection marker for the creation of gene disruption constructs. The target genes were subcloned into pUC119, cut at a unique internal restriction site, blunt-ended, and ligated with the end-blunted selection marker. Tie inserts were then transferred from pUC119 to pKC1139, and conjugated into wild-type S. lavendulae. Transconjugants were selected on AS1 plates (Baltz, 1980), overlaid with apramycin, kanamycin, and nalidixic acid followed by propagation on R5T plates (g/L: sucrose 121.1, K₂SO₄ 0.3, MgCl₂. 6H₂O 11.92, glucose 11.8, yeast extract 5.89, casamino acids 0.12, trace elements 2.35 ml (Hopwood et al., 1985), agar 25.9, after autoclaving the following solutions were added: 0.5% KH₂PO₄ 11.8 ml, 5 M CaCl₂ 4.71 ml, 1 N NaOH 8.25 ml) at 37° C. for several generations. Disruption mutants were selected based on the phenotype changing from apramycin and kanamycin resistant to apramycin sensitive and kanamycin resistant. Replacement of the chromosomal copy of the target gene with the dismpted plasmid-born copy was confirmed by Southern blot hybridization.

[0221] Mitomycin C Analysis

[0222] MC production was evaluated using 3-day cultures in Nishikohri media (Nishikohri and Fukui, 1978). The culture broth was extracted twice with equal volumes of ethyl acetate. After removing the chemical solvent by vacuum, the crude broth extract was dissolved in 50% methanol and 50% 50 mM pH 7.2 Tris buffer and monitored by HPLC (C₁₈ reverse phase column) at 363 nm. A continuous methanol gradient from 20% to 60% in methanol/50 mM pH 7.2 Tris buffer system over 24 minutes was employed to resolve MC from other crude extract components. A 90% CHCl₃/10% MeOH solvent system was used to resolve and detect MC on TLC plates.

[0223] Results

[0224] Identification of the Mitomycin Biosynthetic Gene Cluster

[0225] The mitomycin cluster was identified by linkage of a cosmid clone containing mrd and a gene (mitA) that hybridized with the rifK gene encoding the rifamycin AHBA synthase (Kim et al., 1998) from Amycolatopsis mediterranei. mitA was subsequently shown to be essential for mitomycin biosynthesis since genetic disruption of the chromosomal copy blocked MC production, and could be complemented with exogenous AHBA (Example 2). Linkage of mitA with one of the mitomycin resistance genes (mrd) implied that the corresponding biosynthetic genes were adjacent to mitA. Cosmid walking was used to obtain overlapping DNA fragments spanning more than 120 kb of the S. lavendulae chromosome adjacent to mitA. Subsequent nucleotide sequence analysis included 55 kb of contiguous DNA, revealing 47 genes involved in mitomycin assembly, regulation and resistance (FIGS. 2 and 5). TABLE 1 MC production in wild-type S. lavendulae and gene disruption mutants No. gene disrupted MC production 0.0 Wild-type control ++ 0.1 additional copy ++ of orf1 in wild-type 1 orf8 ++ 2 orf4 ++ 3 orf1 ++ 4 mitR + 5 mitM − 6 mitI − 7 mitH − 8 mitE − 9 mitB − 10 mitA − 11 mmcA − 12 mmcB − 13 mmcM ++ 14 mmcP − 15 mmcR − 16 mmcT − 17 mmcW ++++ 18 mmcX ++++ 19 orf11 ++ 20 orf12 ++ 21 orf16 ++ 22 orf19 ++

[0226] mitT Defines the Left-band Boundary of the Mitomycin Cluster

[0227] Nucleotide sequence analysis extended 30 kb downstream of mitA and revealed a set of genes corresponding to a type I polyketide synthase (PKS, orf9, SEQ ID NO:18; orf8, SEQ ID NO:19) and thioesterase (TEII, orf7, SEQ ID NO:20). MC is not derived from the polyketide pathway, and thusan orf8 disruption mutant showed normal MC production as expected (Table 1). Approximately 20 kb downstream of mitA, two genes (mitT, SEQ ID NO:29 and mitS, SEQ ID NO:30) encoding a putative aminoquinate dehydrogenase and glucose kinase, respectively, were located Both are believed to be involved in AHBA biosynthesis since their equivalents are also present in the rifamnycin biosynthetic gene cluster (rif cluster) (August et al., 1998). However, whether the six genes between orf7 and mitT are involved in MC biosynthesis remained unclear, since the two putative hydroxylases (orf3, SEQ ID NO:24 and orf4, SEQ ID NO:22) and the candidate activator gene (orf1, SEQ ID NO:26) could play a role in MC production. Both orf3 and orf4 are predicted to encode cytochrome P450 monooxygenases with Orf4 most similar to OleP and RapN (50% identity, 63% similarity) for oleandomycin and rapamycin biosynthesis, respectively (Rodriguez et al., 1995; Schwecke et al., 1995). Orf3 shows a high degree of similarity to cytochrome P450 105C1 (49% identity, 64% similarity) in Streptomyces sp. and cytochrome P450-SU2 in Streptomyces griseolus (Horii et al., 1990; Omer et al., 1990).

[0228] Database analysis revealed that Orf1 belonged to the ActII-ORF4, RedD, DnrI and CcaR family of Strepiomyces antibiotic pathway specific activators regulating the production of actinorhodin, undecylprodigiosin, daunorubicin, and cephamycin, respectively (Fernandez-Moreno et al., 1991; Perez-Laraine et al., 1997; Takano et al., 1992; Tang et al., 1996; Wietzorrek and Bibb; 1997). A common feature of this group of activators is that disruption of the corresponding gene abolishes the production of the corresponding antibiotic while overexpression results in a several-fold increase in metabolite production. However, when orf1 was disrupted, the mutant strain showed normal MC production (Table 1). Moreover, the wild-type MC producer containing an additional copy of orf1 in pKC1139 also had a normal MC production profile (Table 1). Interestingly, orf4, one of the cytochrome P450 monooxygenase encoding genes adjacent to orf1 also showed normal MC production when disrupted (Table 1). Thus, mitT appears to map to the left-hand end of the mitomycin cluster, while orf1 to orf9 presumably specify biosynthesis of a polyketide product.

[0229] mmcY Defines the Right-hand Boundary of the Mitomycin Cluster

[0230] Nucleotide sequence analysis of the mitomycin biosynthetic gene cluster extended 30 kb upstream of mitA and several orfs corresponding to genes involved in sugar metabolism were identified. They included an acid trehalase (orf12, SEQ ID NO:28), one ABC type transporter (orf16, SEQ ID NO:79), and four adjacent α-amnylases (orf19, SEQ ID NO:82; orf20, SEQ ID NO:83; orf21, SEQ ID NO:84; orf22, SEQ ID NO:85) for starch degradation spanning more than 18 kb (FIG. 2). Disruption of four genes (orf11, SEQ ID NO:27; orf12, SEQ ID NO:28; orf16, SEQ ID NO:79; orf19, SEQ ID NO:82) within this region resulted in mutants with wild-type level MC production profiles, indicating that they fall outside of the mitomycin cluster (Table 1). At the beginning of this group of sugar metabolism genes, a gene (mmcY, SEQ ID NO:75) encoding a presumed chitinase is proposed to be the upstream terminus of the mitomycin cluster. This is evident because mitomycin requires D-glucosamine as a biosynthetic precursor, and MmcY shows 75% identity (85% similarity) with the chitinase C gene (chiC) product from S. griseus that generates N-acetylglucosamine from chitin (Ohno et al., 1996). In addition, mutants with disrupted orf11 and orf12 genes had no effect on MC production, while disruption of mmcW(SEQ ID NO:71) and mmcX(SEQ ID NO:72) both affected MC production significantly (Table 1).

[0231] Mitomycin Resistance Genes

[0232] Antibiotic biosynthetic gene clusters typically include one or more genes for cellular self-protection (Seno and Baltz, 1989). Previous work has identified two mitomycin C resistance genes (mcr and mrd) with mrd linked to mitA (August et al., 1994; Sheldon et al., 1997; Example 2). Subsequent analysis showed that MRD is a resistance protein that binds mitomycin C with 1:1 stoichiometry (Sheldon et al., 1997). However, this resistance mechanism would be extremely inefficient unless the bound drug is transported out of the cell. Indeed, 5 kb upstream of mrd, the mct gene (SEQ ID NO:16, putative mitomycin translocase) encoding a presumed antibiotic transporter was found and shown to be a third resistance component (Example 3). mct encodes 484 amino-acid protein with 14 predicted transmembrane domains. Disruption of mct resulted in a mutant S. lavendulae strain substantially more sensitive to MC, while coexpression of mct with mrd in E. coli dramatically increased MC resistance levels compared to individual expression of the genes (Example 3). In contrast, the high-level MC resistance gene (mcrA) that encodes an MC oxidase (MCRA) capable of reoxidizing activated MC (Johnson et al., 1997) is not linked with this cluster (August et al., 1990; Example 2). Interestingly, database searches identified two McrA homologues (MitR, MmcM) within the MC cluster, both of which encode putative flavoproteins conserved in the FMN/FAD binding motif. MitR displayed weak similarity with McrA (26% identity, 33% similarity), while MmcM showed end-to-end (54% identity, 69% similarity) alignment with the protein. mitR (SEQ ID NO:31) and mmcM(SEQ ID NO:61) were genetically disrupted giving substantially decreased MC production in the mitR mutant strain, in contrast to the mmcM mutant which displayed wild type MC production levels (Table 1).

[0233] Regulatory Genes

[0234] Two genes, mitQ (SEQ ID NO:32) and mmcW(SEQ ID NO:71), were identified in the mitomycin cluster and are presumed to be pathway-specific regulators. MitQ belongs to the OmpR-PhoB subfamily of DNA binding regulators in the two-component regulatory system, with the greatest similarity to members of the phosphate assimilation pathway (PhoR-PhoB) (Makino et al., 1986), ferric enterobactin response pair (PfeR-PfeS) (Dean et al., 1996), and one histidine protein linase—response regulator system (HpkA-DrrA) from Thermotoga maritima (Lee and Stock, 1996). In contrast to the MitQ group of regulators that typically serve as transcriptional activators (Mizuno and Tanaka, 1997), MmcW showed high sequence similarity with the MarR groups of repressors. The most significant similarity corresponds to EmrR, the negative regulator of the E. coli multidrug resistance pump EmrAB (Lomovskaya et al., 1995), and Pacs, a repressor for pectinase, cellulase, and blue pigment production in Erwinia chrysanthemi (Praillet et al., 1996). Significantly, the mmcW disruption mutant displayed a several-fold increase in MC production (Table 1).

[0235] AHBA Biosynthetic Genes

[0236] Precursor incorporation studies previously demonstrated that AHBA is an intermediate for both the ansamycin and mitomycin natural products (Becker et al., 1983; Example 2). Combining the biochemical, enzymatic and molecular genetic results on the biosynthesis of the ansamycin antibiotic rifamycin, Floss has proposed that AHBA is derived from the ammoniated shikimate pathway via phosphenolpyruvate (PEP) and erythose 4-phosphate (E4P) by the early incorporation of nitrogen (Kim et al., 1996). In the shikimate pathway, PEP and E4P is first converted to 3deoxy-D-arabino-heptulosonic acid-7-phosphate (DBAHP) then stepwise transformed to 3dehydroquinate (DHQ), 3-dehydroshikimate (DHS) and shikimate, catalyzed by DAHP synthase, DHQ synthase, DHQ dehydratase, and shikimate dehydrogenase, respectively (Dewick, 1998). Quinate can also enter the pathway by the action of quinate dehydrogenase to generate DHQ.

[0237] Evidence to support this new variant of the shikimate pathway includes the following experimental observations. First, all proposed ammoniated shikimate pathway compounds including PEP, E4P, 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP), 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) can be readily converted into AHBA by cell-free extracts from the ansamycin producers, while none of the early shikimate pathway intermediates, DAHP, DHQ, DHS, quinic acid, shikimic acid can be incorporated into AHBA under the same conditions (Homemann, 1981; Kim et al., 1996). Second, the rifamycin biosynthetic gene cluster (rif cluster) has been sequenced, and all of the genes encoding early shikinmate pathway enzymes are found within the cluster (August et al., 1998). Finally, the ability of the rifamycin AHBA synthase (RifK) to catalyze dehydration of aminoDHS to AHBA has been previously demonstrated (Kim et al., 1998). As described in Example 2, the AHBA synthase gene (mitA) in S. lavendulae is required for AHBA biosynthesis.

[0238] A group of AHBA biosynthetic genes similar to those described for nf have been identified in the mitomycin cluster. In addition to AHBA synthase, six gene products in the cluster showed high sequence similarity (over 43% identity) with their rifamycin AMBA biosynthetic gene homologs. These gene products include aminoDHQ synthase (MitP, RifG equivalent), aminoquinate dehydrogenase (MitT, RifI equivalent), oxidoreductase (MitG, RifL, equivalent), phosphatase MitJ, RifM equivalent), kinase (MitS, RifN equivalent), and aminoDHQ dehydratase (MmcF, RifJ equivalent). In addition to the significant sequence similarity to rifamycin counterparts, all three putative mitomycin shikimate pathway enzymes displayed significant alignment with microbial primary shikimate metabolic enzymes including MitT with the quinate dehydrogenase (AroE) from Methanococcus jannaschii (28% identity, 46% similarity) (Bult et al., 1996), MitP with the DHQ synthase (AroB) from Mycobacterium tuberculosis (46% identity, 61% similarity) (Cole et al., 1998), and MmcF with the DHQ dehydratase from S. coelicolor (50% identity, 62% similarity) (White et al., 1990). Despite extensive sequencing of 15 kb on either side of the mapped right- and left-hand ends of the mitomycin cluster, an aminoDAHP synthase gene corresponding to RifH (the proposed first enzyme in the de novo biosynthesis from PEP and E4P in the rif cluster), was not found (FIG. 2). Interestingly, a rifH homologue has been cloned from S. lavendulae genomic DNA through Southern hybridization and shown to be unlinked to the mitomycin cluster.

[0239] The existence of non-shikimate pathway-related phosphataselkinase pair in the mitomycin cluster (MitJ/MitS) and the rif cluster (RifM/RifN) further support the finding that these two genes are required for AHBA biosynthesis (Floss, 1997). In addition to the strong homology to RifM, MitJ also showed 56% identity (69% similarity) with ORF8 from the ansamycin antibiotic ansamitocin producer Actinosynnema pretiosum auranticum. Other polypeptides with considerable sequence similarity belong to the CBBY family of phosphoglycolate phosphatases in glycolate oxidation (Schaferjohann et al., 1993). MitS, most similar to RifN (53% identity, 63% similarity), also showed significant similarity with the glucose kinase (involved in glucose repression) from S. coelicolor and Bacillus megaterium (Angell et al., 1992; Spath et al., 1997). mitG, the third non-shilimate pathway-related AHBA biosynthetic gene in this cluster is also worthy of note since it shows exclusive similarity (46% identity, 61% similarity) with oxidoreductase RifL, and its equivalent in Actinosynnema pretiosum auranticum.

[0240] Mitosane Fonnation Genes

[0241] Precursor incorporation studies established that the mitosane core is assembled form the condensation of AHBA and D-glucosamine. Although no specific gene products can be assigned for forming the three bonds bridging AHBA and D-glucosamine, two genes downstream of mitA (SEQ ID NO:97), mitB (SEQ ID NO:99), and mitE (SEQ ID NO:44) likely encode enzymes that mediate one of these reactions. MitB shows local sequence similarity with a group of glycosyltransferases involved in glycopeptide antibiotic and polysaccharide biosynthesis, the typical function of which is to attach an activated sugar residue to a core compound (Yamazaki et al., 1996,Example 2). Meanwhile, MitE showed weak similarity (22% identity and 45% similarity) to the two cloned 4-hydroxybenzoate-CoA ligases from Rhodopseudomonas palustris in the anaerobic degradation of aromatic compounds (Gibson et al., 1994). It also showed similarity to a group of long chain fatty acid CoA ligases, as well as to the O-succinylbenzoic acid CoA synthetase in Vitamin K2 biosynthesis (Kwon et al., 1996). mitB and mitE disruption mutants both had a MC deficient phenotype (Table 1).

[0242] The condensation of AHBA with D-glucosamine may be initiated in two different ways. This includes either initial formation of a C_(8a)-C₉ bond by an acylation or alkylation reaction, or formation of a Schiff base between the MABA nitrogen and D-glucosamine C1 aldehyde, followed by the ring closure at C_(8a)-C₉. mitR (SEQ ID NO:31), one of the two McrA homologues may be involved in one of the ring closure reactions. Interesting, MitR showed high sequence homology with the plant berberine bridge enzyme (BBE) (30% identity, 37% similarity) in benzophenanthridine alkaloid formation, where it catalyzes an unusual C-C bond formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline (Dittrich and Kutchan, 1991). Using a mechanism similar to BBE, it is possible that MitB is involved in C_(8a)-C₉ bond formation. The decreased MC production in the mitR disruption mutant may be due to the existence of isoenzymes (e.g., MmcM) that could catalyze the reaction in the absence of a functional MitR

[0243] Side Group Modification Genes

[0244] Complete assembly of MC requires functionalization of several sites on the core mitosane ring system. First, complete reduction of the carbonyl group at C-6 must occur. Second, hydroxylation at C-5 and C9a must proceed followed by methylation at C-9a. Third, amination at C-7 must occur presumably through initial hydroxylation followed by transamination. Fourth, oxidation of the hydroxyl groups at C-5 and C-8 to form the benzoquinone are required. Fifth, intramolecular amination of C-1 by N-1a to form the aziridine ring must be completed and finally, carbamoylation at C-10 completes assembly of the molecule. Several enzymes found in this cluster likely catalyze these modifications and are discussed below.

[0245] Methylation

[0246] In contrast to MC which has an O-methyl group at C-9a, mitomycin A and mitomycin B also contain a C-7 O-methyl group, while mitomycin B, mitomycin D and porfiromycin have an N-methyl on the aziridine ring (FIG. 1). Radio-labeled precursor incorporation studies showed that all of the O- and N-methyl (but not the C-methyl) groups in the mitomycin molecules are derived from L-methionine (Bezanson and Vining, 1971). Typically, the methyl donor for most C1 reactions is S-adenosyl-L-methionine (SAM), which can be formed through activation of L-methionine by ATP. Three SAM dependent methyltransferase genes were identified in this cluster (encoding MitM, MitN, and MmcR), all of which have three conserved S-adenosylmethionine or S-adenosylhomocysteine binding motifs (Kagan and Clarke, 1994) (FIG. 3). Interestingly, database searches of MitM and MitN (likely responsible for the MC C-9a side chain methylation) revealed a group of plant δ-(24)-sterol C-methyltransferases that have a closer phylogenetic relationship with the rifarnycin O-methyltransferase (ORF14) and erythromycin O-methyltransferase (EryG) (5, 86) (FIG. 4). In contrast, protein database searches revealed that MmcR is most related to other Streptomyces antibiotic biosynthetic O-methyltransferases with greatest similarity to O-demethylpuromycin O-methyltransferase (44% identity, 60% similarity) from S. anulatus and carminomycin 4-O-methyltransferase from S. peucetius (Lacalle et al., 1991; Madduri et al., 1963). MmcR may be involved in the O-methylation of the phenol ring of MC before oxidation to the quinone. Both mmcR (SEQ ID NO:67) and mitM (SEQ ID NO:36) were shown to be essential for MC biosynthesis since disruption of each one completely abolished MC production (Table 1).

[0247] A SAM-independent methyltransferase, MmcD, was also identified in the mitomycin cluster. MmcD revealed strong sequence homology with the magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (34% identity, 53% similarity) from Methanobacterium thermoautotrophicum (Accession Number 2622915), as well as the phosphonoacetaldehyde methyltransferase from Streptomyces wedmorensis (Hidaka et al., 1995), the P-methyltransferase from Streptomyces hygroscopicus (Hidaka et al., 1995) and the fortimicin KL methyltransferase from Micromonospora olivasterospora (Kuzuyama et al., 1995). Instead of SAM, this group of methyltransferases uses methylcobalamine or a structurally related protoporphyrin as the direct methyl donor. While the greatest number of matches were made to protoporphyrin methyltransferases, it is expected that this enzyme has another function in the mitomycin C biosynthetic pathway as all the O- and N-methyl groups of MC have been shown to be derived from SAM-dependent methyltransferases.

[0248] C-6 Carbonyl Reduction

[0249] The C-6 methyl group was previously shown to be derived from the reduction of the carboxylic acid of AHBA, since [carboxy-¹³C] AHBA can be efficiently, and specifically incorporated into the C-6 methyl group of porfiromycin (Anderson et al., 1980). In the mitomycin cluster, four F420-dependent tetrahydromethanopterin (H₄MPT) reductase genes (encoding MitH, MitK, MmcI, MmcJ) and one H₄MPT:CoM methyltransferase gene (encoding MmcE) are candidates for the C-6 carbonyl reduction. In the methanogenesis pathway of Methanobacterium thermoautotrophicum, two cofactor F420-dependent H₄MPT reductases, and one cofactor CoM dependent methyltransferase are required in the seven step reduction from CO₂ to CH₄. Steps 4 to 6 from CH-H₄MPT to CH₂-H₄MPT, and CH₃-H₄MPT to CH₃-CoM are catalyzed by N⁵, N¹⁰-methylene-H₄MPT dehydrogenase, N⁵, N¹⁰-methylene-H₄MPT reductase, and N⁵-methyl-H₄MPT:CoM methyltransferase, respectively (Depperuneier et al., 1996; Thauer et al., 1993). All four enzymes (MitH, MitK, MmcI, MmcJ) in this cluster showed local sequence similarity with the cloned F420 dependent H₄MPT reductase (42% identity, 62% similarity in several 50 amino-acid fragments) (Nolling et al., 1995; Vaupel and Thauer 1995). One of these genes, mitH (SEQ ID NO:41) was disrupted, and the mutant strain displayed a MC deficient phenotype (Table 1). MmcE is notable since the deduced protein sequence contains two domains showing significant alignment (33% identity, 56% similarity) to the N-terminus of H₄MPT:CoM methyltransferase from Methanobacterium thermoautotrophicum (Stupperich et al., 1993), while the remaining C-terminus is related to fatty acid biosynthetic acyl carrier proteins (ACP) (Morbidoni et al., 1996; Platt et al., 1990). The potential function of this ACP-like domain in MC biosynthesis remains unknown, as does the role of a distinct gene (mmcB, SEQ ID NO:50) encoding a putative ACP identified just upstream of mmcE (SEQ ID NO:53). Significantly, the disruption of mmcB resulted in total abrogation of MC production (Table 1).

[0250] Hydroxylation

[0251] The two putative hydroxylases (encoded by mmcN, SEQ ID NO:62; and mmcT, SEQ ID NO:69) identified in the mitomycin cluster are candidates for catalyzing hydroxylation at the C-5, C-7, and C-9a positions on the mitosane system. MmcN belongs to the cytochrome P450 family of monooxygenases, with greatest homology (37% identity, 56% similarity) to the two herbicide-inducible cytochrome P450s (P450-SU1 and P450-SU2) from S. griseolus, as well as to RapJ and RapN in the rapamycin biosynthetic gene cluster from S. hygroscopicus (Omer et al., 1990; Schwecke et al., 1995). MmcT showed highest similarity to the tetracenomycin C hydroxylase (TcmG) in Streptomyces glaucescens (38% identity, 55% similarity), with lower but significant sequence similarity to a group of phenol or hydroxybenzoate hydroxylases (Decker et al., 1993). Genetic disruption of mmcT completely blocked MC biosynthesis (Table 1)

[0252] Carbamoylation

[0253] The carbamoyl group of MC is derived intact from L-citrulline or L-arginine with carbamoyl phosphate as the incorporated precursor (Homemann, 1981). In eubacteria, carbamoyl phosphate can be generated from L-glutamine, HCO₃ ⁻, and ATP by the enzyme carbamoyl phosphate synthetase, which is indispensable for pyrimidine biosynthesis. One candidate carbamoyl transferase gene (mmcS, SEQ ID NO:68) was identified directly upstream of mmcT. MmcS belongs to the NodU/CmcH family of O-carbamoylation enzymes, with the greatest similarity (35% identity, 44% similarity) to NolO from Rhizobium sp. (Jabbouri et al., 1998). Other members with significant alignment in this family include NolO from Bradyrhizobium japonicum (Luka et al., 1993) and NodU from Rhizobium sp. for 6-O-carbamoylation of Nod-factors (Jabbouri et al., 1995) and CmcH from Nocardia lactamdurans and S. clavuligerus for 3′-hydroxyrnethylcephem O-carbamoylation in cephamycin biosynthesis (Coque et al., 1995).

[0254] Discussion

[0255] Bridging Primary and Secondary Metabolism

[0256] The shikimate pathway is an essential metabolic route in microorganisms and plants for aromatic amino acid biosynthesis. Genes encoding the early shikimate pathway enzymes from various organisms have been well studied and are often dispersed along the chromosome as revealed by genome sequencing projects (Blattner et al., 1997; Bult et al., 1996; Cole et al., 1998). The finding that the ansamycin and mitomycin natural products are derived in part from an ammoniated shikimate pathway whose genes are clustered on the bacterial chromosome is a significant difference to the primary metabolic network, and may suggest an important evolutionary bridge leading to secondary metabolism. The lack of incorporation of early shikimate pathway intermediates into mitomycin and ansamycin metabolites indicated the existence and ultimate substrate specificity of the alternate ammoniated shikimate pathway enzymes. However, the conversion of aminoDAHP and aminoshikimic acid by the corresponding primary shikimate pathway enzymes to aminoDHQ and aminoDHS, respectively (Kim et al., 1996), suggested that the substrates specificity in primary metabolic shikimate pathway is mainly determined by the initial reaction step. This notion is further supported by the disruption results for rifG and rifI mutants showing only slightly affected rifamycin production (Floss, 1997).

[0257] In addition to the absence of an aminoDAHP synthase gene, the organization of the AMBA biosynthetic genes in the MC cluster is quite different compared to the rif cluster. In rif (with the exception rifJ), all AMBA biosynthetic genes are found within a defined subcluster that are organized into a single apparent operon. In contrast, almost all of the mit/mmc encoded AHBA genes are scattered within the 55 kb MC cluster. Thus, as opposed to the multifunctional polyketide gene clusters whose linearity of architecture reflects a precise pattern of biosynthesis, the MC cluster is biochemically less transparent based on a similar primary analysis. In addition, the MC cluster provides a good model for analyzing genetic evolution both vertically, from the primary metabolic shikimate pathway to the secondary shikimate pathway related route, and horizontally by comparing different groups of secondary metabolic biosynthetic clusters.

[0258] The MC Biosynthetic Network

[0259] In a typical liquid culture of S. lavendulae, MC production initiates 24 hours after inoculating the seed culture, reaches maximum production in two days, and maintains drug synthesis during stationary phase for another two days. Compared to high level MC resistance of the wild-type S. lavendulae (>150 μg(ml), MC production is relatively low (<5 μg/ml MC). The significant gap between the self-resistance and production levels makes it possible to improve drug production through genetic engineering. As described herein, disruption of the candidate repressor gene (mmcW) and downstream mmcX(encoding a putative membrane protein) in the mitomycin cluster resulted in a several-fold increase in MC production. The existence of a repressor gene(s) is not uncommon in Streptomyces antibiotic biosynthetic gene clusters. Previous examples include, mmyR from the methylenomycin cluster (Chater and Briton, 1985), actII-orfI in the actinorhodin cluster (Caballero et al., 1991),jadR (Anderson et al., 1980) in jadomycin biosynthesis (Yang et al., 1995), and dnrO in the daunorubicin cluster (Otten, 1995). Disruption of jadR and mmyR also resulted in increased levels of the corresponding antibiotic (Chater and Briton, 1985; Yang et al., 1995).

[0260] In order to avoid auto-toxicity, drug-producing microorganisms must evolve self-protection systems. Currently, three types of self-protection mechanisms have been identified in S. lavendulae for mitomycin resistance including, MC binding (MRD), efflux (MCT), and reversing MC reductive activation (MCRA). In principle, resistance genes must be expressed before drug formation. In this respect, it is interesting to note the linkage of the mitomycin resistance genes with the regulatory genes. Expression of the high-level resistance gene mcrA has been demonstrated to be regulated by the downstream gene mcrB which is presumably cotranscribed with mcrA (August et al., 1994). Though the function of the McrA homolog MitR in the mitomycin cluster remains unknown, mitR is also followed by a cotranscribed regulatory gene (mitQ). Meanwhile, the putative mitomycin translocase gene, mct is followed by the repressor gene, mmcW. Genetic linkage of membrane transporter/resistance and repressor genes have been described in a number of cases, including tetA/tetR in tetracycline resistance (Guilfoile and Hutchinson, 1992), tcmA/tcmR in tetracenomycin C resistance (Guilfoile and Hutchinson, 1992), actII-orf2/actII-orf1 in actinorhodin resistance (Caballero et al., 1991), and the qacA/qacR pair for multidrug resistance in S. aureus (Grkovic et al., 1998).

[0261] Conclusion

[0262] Although MC was first isolated more than 40 years ago and has been used in anti-cancer chemotherapy since the 1960s, the mechanistic details and order of its biosynthesis has remained unclear. The results described herein are clearly consistent with precursor incorporation studies gathered in the 1970s, showing that MC is biosynthetically derived from D-glucosamine, L-methionine, carbamoyl phosphate, and AHBA, and also support the use of the variant de novo shikimate pathway leading to AHBA (Homemann, 1981; Kim et al., 1996). Many, if not all, of the genes responsible for the formation of the mitosane and aziridine rings are evidently located within the boundary of the 55 kb mitomycin cluster. These genes are of special interest since they may be useful as probes for identification of related natural product biosynthetic genes from other microorganisms and plants.

[0263] The cloned genes presented here are useful to study mitomycin biosynthesis and natural product assembly. The advantage of having this information has already been demonstrated through genetic disruption of the candidate repressor gene (mmcW) that provided a several-fold increase in MC production. In addition, expression and genetic disruption of selected genes should be useful for engineering the biosynthesis of clinically valuable mitomycin analogues, as well as more complex hybrid natural product systems. Finally, the MC resistance and regulatory genes identified in this cluster provide important insight into the mitomycin biosynthetic and regulatory network in the S. lavendulae.

EXAMPLE 2 Genetic Localization and Molecular Characterization of Two Genes Required for MC Biosynthesis

[0264] Materials and Methods

[0265] Strains and culture conditions. E. coli DH5α was grown in either Luria broth (LB) or tryptic soy broth (TSB) (Difco) as liquid medium or agar plates. E. coli DH5αF′, the host for harvesting single-stranded DNA, was grown at 37° C. on TBG (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH₂PO₄, 55 mM K₂HPO₄, and 20 mM glucose). E. coli S17-1 (Mazodier et al., 1989) used for conjugation was grown in TSB with 10 μg/ml of streptomycin. S. lavendulae was grown in TSB or on R5T plates. For MC production, S. lavendulae was grown in Nishikohri media (g/L: glucose 15, soluble starch 5, NaCl 5, CaCO₃ 3, yeast extract 5) for 72 hours from a 1% v/v inoculum of frozen mycelia. Pulse feeding of AHBA to the disruption mutant, MV100, and the site-directed mutant, MV102, occurred with feedings of 2.5 mg of a 20 mg/mL solution of the sodium salt of AHBA at pH 7.1 in three pulses at 24, 43, and 57 hours of a culture that was harvested at 76 hours.

[0266] DNA preparation and amplification. Isolation and purification of DNA was performed using standard methods (Sambrook et al., 1989). S. lavendulae NRRL 2564 genomic DNA was isolated by using the modified Chater protocol (Hopwood et al., 1988). Plasmid DNA was isolated from E. coli by using the alkaline-sodium dodecyl sulfate method.

[0267] pDHS2002 was constructed as follows: The 1.1 kb thiostrepton resistance gene (tsr) fragment was removed from pDHS5000 with a SmaI-BamHI digestion, blunt-ended with the large fragment of DNA polymerase (Gibco BRL), and ligated to MscI restriction enzyme digested pDHS7601 to yield pDHS20001. MscI digestion of pDHS7601 resulted in the removal of 155 nucleotides at the C-terminus of the mitA gene, and ligation of the blunt-ended BamHII site of the tsr adjacent to the MscI site of pDHS7601 resulted in regeneration of the BamHI site in pDHS2001. The 4.9 kb EcoRI-HindIII fragment from pDHS2001 containing the tsr disrupted mitA gene was removed and ligated into EcoRI-HindIII digested pKC1139 to yield pDHS2002.

[0268] Primer-mediated site-directed mutagenesis (SDM) was employed to construct pDHS2015 containing a K191A mutation in mitA. Primer 1: 5′-GGCAAGGCATGCGAGGGTCGC-3′ (SEQ ID NO:46) and primer 2: 5′-TTCCAGAACGGCGCCCTGATGACCGCCGGC-3′ (SEQ ID NO:47) were used to amplify the 691 bp fragment of the 5′ end of mitA. The 3′ end of mitA was amplified with primer 3: 5′-GCCGGCGGTCATCAGGGCGCCGTTCTGGAA-3′ (SEQ ID NO:48) and primer 4:5′-TCAGAATTCGGATCCGAGGGCCGGAGT-3′ (SEQ ID NO:86) to generate a 1151 bp band (see amplification reaction conditions in Example 3). A second round of PCR was performed using the overlapping 691 and 1151 bp units as the initial templates with primer 1 and primer 4 to yield a 1.8 kb fragment. The final product containing mutagenized mitA was digested with EcoRI-Sph1, ligated to the 2.1 kb HindII-SphI fragment from pDHS7601 and the EcoRI-HindIII digested pKC1139 to yield pDSH2015. The site-directed mutation of MitA K191A in pDHS2015 was confirmed by sequencing with forward primer: 5′-ACCTACTGCCTCGATGCC-3′ (SEQ ID NO:87) and reverse primer: 5′-CTGATCCTTCAAGCG-3′ (SEQ ID NO:88).

[0269] The mitB disruption vector pDHS7702 was constructed as follows. pDHS7601 was digested with BstBI, blunt-ended, and ligated with the 1.4 kb neomycin-resistant gene fragment from pFD666 (Denis and Brzezinski et al., 1992) (ApaL1-HindIII digestion, blunt-ended). The 5.2 kb EcoRI-HindIII fragment from the resulting construct pDHS7701 was subeloned into pKC1139 to create pDHS7702.

[0270] DNA library construction and screening. S. lavendulae NRRL 2564 genomic DNA was partially digested with Sau3AI, and a fraction containing 30-50 kb fragments was recovered by sucrose gradient centrifugation and ligated into the calf intestinal alkaline phosphatase (CIP) treated BglII site of the E. coli-Streptomyces shuttle vector pNJ1 (Tuan et al., 1990), then packaged with the Packagene Lambda DNA Packaging System (Promega). The cosmid library was constructed by transfecting E. coli DH5α, and colonies that appeared on the LB plates containing 100 μg/ml of ampicillin were transferred to a BioTrace NT nitrocellulose blotting membrane (Gelman Sciences, Ann Arbor, Mich.). Colony hybridization was performed as specified by the manufacturer. A PCR-amplified 0.7 kb DNA fragment from plasmid pKN108 (FIG. 6) was used to screen the library. The primers used for PCR were: 5′-GCGTCCGTGCTGCGCGCGCA-3′ (SEQ ID NO:89), and 5′-TGCGCGCGCAGCACGGACGC-3′ (SEQ ID NO:90). The cosmids from the positive colonies were confirmed by Southern blot hybridization, and a 1.7 kb AflIII-BamHI fragment from pDHS3001 containing the mitomycin resistance determinant (mrd) (Sheldon et al., 1997) was used as a probe to establish genetic linkage.

[0271] DNA sequencing and analysis. Deletion subclones from pDHS7601 were made with exonuclease III Erase-a-Base System (Promega). Sequencing was accomplished with the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and analyzed on an Applied Biosystems 377 DNA Sequencer at the University of Minnesota Advanced Genetic Analysis Center. For generating single-stranded DNA, deletion subclones in pUC119 were transformed into E. coli DH5αF′, and M13K07 Helper Phage was used (GIBCO BRL). Nucleotide sequence data were analyzed using Wisconsin Genetics Computer Group software (version 9.0) (Devereux et al., 1984), and GeneWorks software version 2.51 (Oxford Molecular Group). The GenBank accession number for mitABC is AF115779.

[0272] Conjugation from E. coli S17-1 to S. lavendule. The procedure of Bierman et al. (Bierman et al., 1992) was used with the following modification. A single colony of E. coli S17-1/pDHS2002 was used to inoculate 2 ml of TSB containing 100 μg/ml of apramycin and 10 μg/ml of streptomycin. Following overnight incubation at 37° C. a 1:100 inoculation was made into TSB broth with 100 μg/ml of apramycin and 10 μg/ml of streptomycin. This culture was grown for 3 hours at 37° C., and the cells were washed twice with TSB and resuspended in 2 ml of TSB to provide the donor E. coli culture. The recipient S. lavendulae culture was generated by inoculating 9 ml of TSB with 1 ml of frozen wild-type culture. Following overnight (16 hour) incubation at 29° C., the culture was homogenized by sonication and 2 ml of this culture was used to inoculate 18 ml of TSB. Following overnight growth at 29° C. and sonication treatment to homogenize the culture, a 1 ml inoculum was placed in 9 ml of TSB. This culture was grown for 3 hours, the mycelia were washed with TSB and resuspended in 2 ml of TSB to provide the stock recipient culture.

[0273] The donor and recipient cultures were mixed together in 9:1, 1:1, and 1:1/10 donor:recipient ratios, and 100 μl of the cell mixture was spread on AS1 plates (Baltz, 1980). The plates were incubated overnight at 29° C. and overlaid with 1 ml of water containing a suspension of 500 μg/ml each of thiostrepton, apramycin and nalidixic acid. For the pKC1139 control, only apramycin and nalidixic acid were overlaid, while for pDHS7702, 500 μg/ml of kanamycin was used instead of thiostrepton. S. lavendulae exconjugates appeared in approximately 11-13 days at a frequency ranging from 10⁻⁷-10⁻⁵. pKC1139 has a temperature-sensitive Streptomyces replication origin, which is unable to replicate at temperatures above 34° C. (Muth et al., 1989), while the S. lavendulae host grows well at 42° C. Thus, after propagating the conjugants at 39° C. for several generations, double crossover mutants were readily generated. Presence of plasmid was determined by transformation of E. coli DH5α with plasmid extracts from S. lavendulae transconjugants.

[0274] Double-crossover selection procedure. A single colony of S. lavendulae/pDHS2002 grown on R5T plates (50 μg/ml of thiostrepton and apramycin) was used to inoculate TSB broth containing 20 μg/ml of thiostrepton. After 72 hours of incubation at 39° C., 10⁻⁴, 10⁻⁵ and 10⁻⁶ diluted aliquots were used to inoculate R5T plates containing 50 μg/ml of thiostrepton. Following 48 hours of growth at 39° C., 84 colonies were picked randomly and each colony was patched out on separate 50 μg/ml of thiostrepton and 50 μg/ml of apramycin containing R5T plates. One of the 84 colonies displayed the double crossover phenotype of thiostrepton resistance and apramycin sensitivity. Integration of the tsr disrupted mitA gene and loss of plasmid pDHS2002 was confirmed by Southern hybridization analysis.

[0275] MitA K191A site-directed mutants (MV102) were selected by propagating MV100/pDHS2015 on R5T plates for two generations at 37° C. Colonies were replicated to plates containing 50 μg/ml of thiostrepton and plates without antibiotics. Of the 108 colonies replicated in the first round, one had the correct (thiostrepton sensitive) phenotype. To confirm the K191A mutation, the mitA gene was amplified from the chromosome with primers 1 and 4. Mutation of the conserved lysine codon (AAG) to an alanine codon (GCC) was verified with the same sequencing primers employed to confirm the correct construction of pDHS2015. The alanine codon was observed in both the forward and reverse sequence data.

[0276] Mutants for mitB (MM101) were selected as follows: S. lavendulae/pDHS7702 was propagated on R5T plates for five generations at 39° C. before single colonies were replicated on R5T plates as described above. Of the 300 colonies tested, 12 clones displayed the correct phenotype (kanamycin resistance and apramycin sensitivity). The genotype of selected mitB mutants was confirmed by Southern blot hybridization of S. lavendulae genomic DNA.

[0277] Analysis of MC production. All cultures intended for MC extraction were grown in Nishikohri media (Nishikohri and Fukui, 1975) for a period of 72 hours. In all cases a wild-type S. lavendulae culture was grown concurrently with the mutant cultures to provide a MC production reference point. A 72 hours, 50 ml culture (250 ml flask) of the MitA K191A MV102 mutant strain was supplemented with 125 μl of a 20 mg/ml solution of the sodium salt of AHBA (pH 7.05) at 24, 43 and 55 hours. In each case, the culture broth was separated from mycelia by centrifugation and then extracted three times with equal volumes of ethyl acetate. The ethyl acetate extracts were pooled and solvent was removed by vacuum to provide the crude broth extract. The preliminary screen for MC production involved thin layer chromatography (TLC) on silica gel plates (Whatman K6) eluted with 9:1 chloroform:methanol. Production of MC was monitored by HPLC (C₁₈ reverse phase column) using a gradient of 80% 50 mM Tris buffer (pH 7.1)/20% methanol to 40% 50 mM Tris buffer (pH 7.2)/60% methanol with the UV detector set to 363 nm.

[0278] Bioassay detection of MC was performed by loading a 1 cm disk with fractions eluting at the mitomycin retention time from HPLC injections of wild-type, MV100, pKC1139 vector control crude extracts and MC standards. The disks were placed on antibiotic media number 2 agar plates (Difco) with Bacillus subtilis spores added directly to the media. The plates were incubated overnight at 29° C. and examined for zones of inhibition. To confirm the production of MC by MV102 in the presence of exogenous AHBA the fraction eluting at the MC retention time was collected, dried down, desalted and submitted for desorption ionization mass spectrometric analysis on a Bio-Ion 20R DS-MS instrument (Applied Biosystems). The MC (M. W.=334)-sodium (M. W.=23) adduct peak, [M+Na]⁺=357, was diagnostic for the presence of MC in the AHBA supplemented culture.

[0279] Results

[0280] The mrd and ahbas genes are linked in the S. lavendulae genome. Southern blot analysis with the A. mediterranei AHBA synthase (rifK) gene probe (Kim et al., 1998) showed a single 3.8 kb band that hybridized with BamHI digested S. lavendulae genomic DNA (FIG. 8). Subsequently, a S. lavendulae genomic DNA library was constructed using the E. coli-Streptomyces shuttle cosmid pNJ1. Of the 5,000 colonies screened, 21 positive clones were identified with six of these hybridizing with the mrd gene probe (none hybridized with the mcr gene probe described in August et al., 1994). Restriction-enzyme mapping and reciprocal hybridization of the cosmid clones established that the mrd and S. mediterranei AHBA synthase homologous genes were about 20 kb apart in the S. lavendulae genome. The 3.8 kb BamHI fragment bearing a putative S. lavendulae AHBA synthase gene was subcloned and its nucleotide sequence determined.

[0281] Three ORFs are identified within the 3.8 kb BamHI fragment. Three ORFs (mitA, mitB, mitC) were identified within the sequenced 3.8 kb BamHI fragment (FIGS. 8 and 9). mitA comprises 1164 nucleotides and starts from ATG (position 579 of the sequenced fragment) that is preceded by a potential ribosome binding site (RBS), GAAAGG (SEQ ID NO:91). The deduced product of the mitA gene encodes a hydrophilic protein of 388 amino acids with a predicted M_(r) of 41,949 Da and a calculated pI of 5.62. A BLAST (Altschul et al., 1990) search showed that the predicted MitA protein has high sequence similarity (about 71% identity, 80% similarity) with AHBA synthases (AHBASs), both from the rifamycin producer A. mediterranei (Kim et al., 1998) and other ansamycin-producing actinomycetes, including Actinosynnema pretiosum (ansamitocin) and Streptomyces collinus (naphthomycin A and ansatrienin) (FIG. 10). A conserved pyridoxal phosphate (PLP) coenzyme binding motif (GX₃DX₇AX₈EDX₁₄GX₁₃KX₄₋₅geGGX₁₉G) (SEQ ID NO:92) including the conserved lysine residue can also be found in these four proteins (Piepersberg, 1994).

[0282] The mitB gene is predicted to start at a GTG (position 1879) that is preceded by a presumed RBS (GGAACG) (SEQ ID NO:93). This gene encodes a 272 amino acid protein with a deduced M_(r) or 28,648 Da and a deduced pI of 6.06. Database sequence homology searches revealed that the product of mitB shows local sequence similarity with a group of O-glycosyltransferases involved in polysaccharide biosynthesis. One segment of 70 amino acid residues at the N-terminus of MitB has 43% similarity (36% identity) with the two glycosyltransferases SpsL and SpsQ from Sphingomonas S88, and ExoO form Rhizobium meliloti involved in polysaccharide (S88) and succinoglycan biosynthesis, respectively (Becker et al., 1963). Another 60 amino acid residues located at the C-terminus displayed 30% identity with UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from Mus musculus and Homo sapiens (Bennett et al., 1996).

[0283] The third ORF, mitC, starts from the ATG at position 2694, which is coupled to the stop codon TGA of mitB and encodes a putative protein of 260 amino acids with a molecular mass of 27,817 Da and a pI of 10.45. Database searches with the deduced protein product showed significant similarity over the first 90 amino acids (38% identity, 40% similarity) with the ImbE gene product (unknown function ) from Mycobacterium leprae (U15183).

[0284] Insertional disruption of the mita and mitB genes in Streptomyces lavendulae. To test the dependence of functional mitA and mitB genes for MC biosynthesis, gene disruption constructs were generated for subsequent isolation of the corresponding S. lavendulae isogenic mutant strains.

[0285] The mitA disruption construct was made by replacing a 155 bp fragment between the two MscI sites (located at the C-terminus of the mitA gene in pDHS7601) with the 1.1 kb SmaI-BamHI fragment containing a thiostrepton resistance gene from pDHS5000 (FIG. 11A). This replacement regenerated a BamHI site at the junction and the resulting construct was then subcloned into the E. coli-Streptomyces conjugative shuttle plasmid pKC1139, followed by conjugation into S. lavendulae. A double crossover mutant strain (MV100) was selected based on the expected phenotype (thiostrepton resistant, apramycin sensitive), and further confirmed by Southern blot hybridization. Genomic DNA from wild-type S. lavendulae and MV100 was digested with BamHI and SphI, and hybridized with the 4.9 kb EcoRI-HindIII tsr-disrupted mitA fragment from pDHS2001. As expected, the 4.0 kb SphI hybridized band in the wild-type strain was shifted to 4.9 kb in MV100, whereas the 3.8 kb BamHI hybridization and in the wild-type was converted to two bands (2.2 kb and 2.5 kb) in the mutant (FIG. 11B).

[0286] The mitB gene was disrupted by inserting a neomycin resistance gene (aphII) into the BstBI site (located at the 5′-end of mitB) (FIG. 12A). Transconjugants were selected on kanamycin/apramycin plates, and a double crossover mutant strain (MM101) was identified with a kanamycin-resistant, apramycin-sensitive phenotype and subsequently confirmed by Southern blot hybridization. As expected, the 3.8 kb BamHI hybridization band in wild-type S. lavendulae was shifted to 5.2 kb in MM101, whereas a 5.2 kb SacI hybridization band was shifted to 6.6 kb (FIG. 12B).

[0287] mitA and mitB disrupted strains (MV100, MM101) are blocked in MC biosynthesis. The growth characteristics and morphology of MV100 and MM1001 in liquid media and on agar plates was identical to wild-type S. lavendulae. HPLC was used to quantify production of MC in MV100 and MM101 (FIG. 13A), and culture extracts were used in a biological assay to test for presence of the drug (FIG. 13B). Injection of one mg of wild-type S. lavendulae culture extract gave a peak in the HPLC that eluted with the same retention time as the MC standard. Upon injection of one mg of culture extract from the mitA or mitB disrupted strains (MV100, MM101) no MC peak was observed. To corroborate the lack of production of MC, the HPLC eluant obtained from the MV100 culture extracts was collected over the retention time range determined for MC. This eluant completely lacked biological activity against Bacillus subtilis (the MC target strain) while the fraction collected from the same retention time region of wild-type S. lavendulae and the vector control strain culture extracts showed substantial levels of biological activity (FIG. 13B).

[0288] It is important to note that the presence of the vector pKC1139 in S. lavendulae reduced the percentage of MC in the total crude extract while simultaneously increasing the total amount of material extractable by ethyl acetate. The combination of these two effects reduces the absolute amount of MC by approximately 25% in the vector control culture crude extract compared to the wild-type crude extract.

[0289] Exogenous AHBA can restore MC, production in the MC-deficient MitA K191A mutant. Although complementation of MV100 (mitA insertional disruptant) was attempted by providing exogenous 3-amino-5-hydroxybenzoic acid in the culture medium, MC production was not restored as measured by HPLC or biological assay. A polar effect on genes downstream of tsr-disrupted mitA in MV100 appeared likely since supplying mitA in trans on a medium copy number plasmid (MV103) also failed to restore MC production. Therefore, site-directed mutagenesis was employed to generate a MitA K191A mutant resulting in strain MV102. Kim et al. (1998) had demonstrated that the AHBA synthase from A. mediterranei is PLP dependent and catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS). Thus, the nitrogen of the conserved lysine 191 is supposed to form a Schiff base with the PLP cofactor. Replacement of lysine 191 with alanine prevents binding of the cofactor and eliminates enzymatic activity. Replacement of the AGG encoding lysine 191 in wild-type S. lavendulae with a GCC codon in MV102 was confirmed by nucleotide sequence analysis. As expected, MV102 did not produce MC, however, when the culture medium was supplemented with exogenous AHBA, MC production was restored as determined by MS ([M+Na]⁺=357), HPLC and TLC analysis (Table 2). TABLE 2 Complementation results with (+) or without (−) AHBA. S. lavendulae MC production strains −AHBA +AHBA Wild-type + + MV100 − − MV103 − − MV102 − +

[0290] Discussion

[0291] An effective strategy for the identification of natural product biosynthetic gene clusters in actinomycetes has included cloning of antibiotic resistance genes followed by investigation of adjacent DNA for the presence of structural and regulatory genes (Butler et al., 1989, Donadio et al., 1991; Motamedi and Hutchinson, 1987; Vara et al., 1985). Although linkage of antibiotic resistance and biosynthetic genes appears to be a general feature in prokaryotes, a growing number of examples involve the existence of multiple resistance loci that may be linked or unlinked to the biosynthetic gene cluster (Vara et al., 1985; Seno and Baltz, 1989; Smith et al., 1995). The identification and characterization of two genetically unlinked resistance loci (August et al, 1994; Sheldon et al., 1997) for MC created a dilemma for mounting an effective search for the MC biosynthetic gene cluster. However, the use of the AHBA synthase gene from A. mediterranei provided an effective probe to identify cosmid clones bearing a linked MC resistance gene. Thus, the isolation of several cosmid clones form an S. lavendulae genomic DNA library that hybridized to both the A. mediterranei AHBA synthase gene and the S. lavendulae mrd gene indicated that the MC biosynthetic gene cluster resided on DNA adjacent to mrd. DNA sequence analysis of the 3.8 kb BamHI fragment revealed three ORFs whose deduced protein sequences corresponded to an AHBA synthase, a glycosyltransferase, and a lmbE-like product.

[0292] As determined by precursor feeding experiments, the mitosane core is formed through the condensation of AHBA and D-glucosamine (Homemann, 1981). AHBA is thought to be derived from the ammoniated shikimate pathway from PEP and E4P, in which the last step from aminoDHS to ABBA is catalyzed by AHBA synthase (FIG. 7) (Kim et al., 1996; Kim et al., 1998). Meanwhile, the reaction of attaching an activated sugar residue to a core compound is usually catalyzed by a group of enzymes called glycosyltransferases as specified by macrolide, glycopeptide antibiotic and polysaccharide biosynthesis (Kahler et al., 1996; Otten et al., 1995b; Solenberg et al., 1997; Yamazaki et al., 1996). In principle, the condensation of AHBA with D-glucosamine can be initiated in two different ways (FIG. 7). One would involve the formation of the C_(8a)-C₉ bond by an electrophilic aromatic alkylation or acylation. A second possibility would be formation of a Schiff base between the nitrogen of AHBA and the D-glucosamine C1 aldehyde, followed by ring closure at C_(8a)-C₉. In either case, a C- or N- instead of O-glycosyltransferase is expected. Although previously described glycosyltransferases display a high degree of sequence divergence (Yamaazaki et al., 1996), the mechanistic similarity with O-glycosyl transfer may suggest that mitB encodes a N-glycosyltransferase that initiates the formation of the mitosane system by linking glucosamine to AHBA. The mitA and mitB genes and their corresponding products are likely candidates to mediate formation of AHBA and the mitosane ring system, respectively. However, the possible function of the lmbE-like protein remains unclear, since its current role within lincomycin biosynthetic pathway of S. lincolnensis is not known (Peschke, 1995).

[0293] The involvement of AHBA synthase (mitA) and the putative glycosyltransferase (mitB) in MC biosynthesis was established by gene disruption to create mutants blocked in MC biosynthesis. This required development of a method to introduce DNA into S. lavendulae NRRL 2564 since the strain remains refractory to traditional Streptomyces protoplast and electroporation-mediated transformation procedures. Other such refractory strains include, but are not limited to, ATCC 27422. The modified Bierman protocol (Bierman et al., 1992) was used to affect efficient conjugative transfer into S. lavendulae using the E. coli-Streptomyces shuttle plasmid pKC1139. This result is significant because it permits the development of an effective system for analyzing in detail the genes involved in mitomycin biosynthesis.

[0294] The function of mitA was probed by providing strains MV100 and MV102 with exogenous 3-amino-5-hydroxybenzoic acid in the culture medium. Despite repeated attempts to complement MV100, MC production was not restored as measured by HPLC or biological assay. It is believed that insertion of the tsr gene into mitA resulted in disruption of biosynthetic genes immediately downstream, since supplying mitA in trans on a medium copy number plasmid also failed to restore MC production to MV100. This putative polar effect was eliminated by generating the MitA K191A mutant strain MV102. Providing exogenous 3-amino-5-hydroxybenzoic acid to this mutant strain of S. lavendulae restored production of MC as shown by TLC, HPLC and mass spectrometry. When MV102 was grown in the absence of AHBA, there was no detectable production of MC. The ability of 3-amino-5-hydroxybenzoic acid to complement the mutant MitA protein further supports the function of MitA as an AHBA synthase as indicated by the database protein sequence alignment and previous studies on rifK (Kim et al., 1998).

EXAMPLE 3 Mitomycin Resistance in Streptomyces lavendulae Includes a Drug-Binding Protein-Dependent Export System

[0295] As a prodrug, MC is unreactive until chemical or enzymatic reduction renders the molecule a highly effective alkylating agent (Iyer and Szybalski, 1964). The molecular basis of MC bioactivity derives mainly from its propensity to covalently interact with DNA at 5′-CpG sequences, causing lethal intra- and inter-strand crosslinks as well as monofunctional alkylation (Tomasz, 1995).

[0296]S. lavendulae encounters a daunting challenge in avoiding potentially lethal MC-mediated crosslinks since it has a chromosomal G+C content of over 70%, which translates into at least one million potential drug target sites per cell. Indeed, two genetic loci that mediate mitomycin resistance have been reported in this organism. One locus (mcr) encodes a protein (MCRA) that catalyzes oxidation of the reduced, bioactivated species of MC via a redox relay mechanism (August et al., 1994; Johnson et al., 1997). The second locus (mrd) encodes MRD that functions to sequester the prodrug by a specific mitomycin-binding protein (Sheldon et al., 1997). A paradox of current knowledge regarding mitomycin resistance has been the lack of a clear mechanism for drug transport. Indeed, the observed stoichiometry suggests that it would be ineffective for S. lavendulae to utilize MRD as a solo mechanism for cellular self-protection. Pathogenic bacteria (Nikaido, 1994), and antibiotic-producing microorganisms (Cundliffe, 1992; Mendez and Salas, 1998), employ export of toxic compounds as a means of resistance.

[0297] Materials and Methods

[0298] Bacterial strains, culture conditions, and media. E. coli DH5α used as a host for generation of double-stranded plasmid DNA, was grown at 37° C. on LB medium. E. coli BL21 (DE3), used as host for protein expression, was grown at 37° C. in NZCYM medium (Sambrook et al., 1989). S. lavendulae NRRL 2564 was grown on YEME medium (Hopwood et al., 1985) at 30° C. for preparation of genomic DNA.

[0299] DNA preparation and amplification. S. lavendulae genomic DNA was isolated by the lysozyme-2×Kirby mix method (Hopwood et al., 1988). General DNA manipulation was performed as described previously (August et al., 1994). Oligonucleotides for PCR and sequencing were obtained from Gibco BRL. PCR amplifications were carried out using a Hybaid thermal cycler (Hybaid Ltd., Teddington, U.K.).

[0300] Cloning and sequencing of mct. A S. lavendulae NRRL 2564 genomic DNA library was constructed in the cosmid vector pNJ1 (Tuan et al., 1990) as previously described (August et al., 1994). The insert DNA of a cosmid clone containing sequences flanking mrd was digested with BamHI and subcloned into the BamHI site of pUC119. Using exonuclease III (Erase-A-Base kit, Promega, Madison, Wis.), a set of nested deletion clones was generated and both strands of the insert DNA were sequenced by the dideoxy chain termination method using the ABI Prism kit (PE Applied Biosystems) in coordination with an ABI 373 automated sequencer. 10% DMSO was added to the reactions to reduce compressions Sequence data was analyzed using the GeneWorks (Oxford Molecular) software package. Deduced amino acid sequence data were compared to the available databases using the BLAST program of the Genetics Computer Group version 9.0 software (Oxford Molecular Group). The mct gene has been deposited in the GenBank database under Accession No. AF120930.

[0301] Construction of the mct mutant strain of S. lavendulae. The mct disruption vector pDHS7704 was constructed as follows. pDHS7661 was digested with EcoRI, blunt-ended, and ligated with the 1.4 kb neomycin resistance gene fragment from pFD666 (ApaLI-HindIII digestion, blunt-ended) (Ames, 1986). The 5.4 kb EcoRI-HindIII fragment from the resulting construct (pDHS7703) was subcloned into pKC1139 to create pDHS7704, and conjugated into S. Iavendulae according to Bierman et al. (1992). A mct double crossover mutant was selected after propagating transconjugants on R5T plates for five generations at 39° C. Kanamycin-resistant and apramycin-sensitive colonies were further tested by Southern blot to confirm the desired double crossover genotype.

[0302] Construction of mct expression plasmid. For the construction of the E. coli expression plasmid NdeI and HindIII sites were introduced at the translational start codon and downstream of the translational stop codon of mct, respectively. The primers used for PCR were 5′-GGGAATTCCATATGATGCAGTCCATGTCAC-3′ (SEQ ID NO:94) and 5′-GGGAATTCAAGCTTTCATTCCGCCGGGGTC-3′ (SEQ ID NO:95). The PCR was carried out using 2.5 U of Taq polymerase, 0.4 μg of each primer, 1 μg of pDHS7661 DNA as template, 10 mM each of dATP-dGTP-dCTP-dTTP, 1.5 mM MgCl₂, and 10 μl of 10×Promega PCR buffer in a total volume of 100 μl . Amplification was achieved with 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 37° C. for 1 minute, and extension at 70° C. for 2 minutes. The 1.45 kb PCR product was recovered by 0.8% agarose gel electrophoresis, digested with NdeI-HindIII and ligated into the T7 expression plasmid pET17b (Novagen), which had been similarly cut with EcoRI-HindIII, to give pDHS7023. pDHS7023 was introduced by transformation into E. coli BL21(DE3) to provide strain PJS102.

[0303] Construction of mct-mrd co-expression plasmid. From plasmid pDHS7006 (Sheldon et al., 1997), a 2.1 kb SspI fragment was isolated. The fragment contained the mrd gene under the control of the T7 promoter, including transcriptional terminator sequences (rrnB T1) upstream and downstream of mrd. The fragment was ligated into the MC-translocase construct pDHS7023, which had been cut with MscI, to give pDHS7024. pDHS7024 was introduced by transformation into E. coli BL21(DE3) to result in strain PJS103.

[0304] MC resistance phenotppe of E. coli. To analyze resistance conferred by the expression of the MC-translocase in E. coli, 10 μl of strain PJS102 was spread on LB agar medium containing 100 μg/ml of ampicillin, IPTG to a final concentration of 1.0 mM, and various concentrations of MC. The cultures were grown overnight at 37° C. and colony-forming units (CFUs) were determined. Similarly, the MC resistance phenotype of strain PJS103 (mcr-mrd co-expression strain) was quantified.

[0305] [³H]-MC uptake assay of strains PJS102 and PJS103. [³H]-MC was obtained from Kyowa Hakko Kogyo, Ltd. Uptake studies were performed for whole cells of PJS100, PJS102, PJS103 and E. coli BL21 (DE3)::pT7SC and pET17b. PJS100, PJS102, and PJS103 as well as vectoronly cultures were cultured (37° C.) in 5 ml of NCZYM medium with IPTG added to a final concentration of 1 mM (at approximately 3 hours growth). At 9 hours (late exponential phase), cells were harvested by centrifugation and resuspended in 1 ml NCZYM broth (5×concentration). The concentrated suspension of late-exponential growth phase cells was exposed to [³H]-MC (59 Ci/mmol) at a final concentration of 0.022 μg/ml (0.0655 nmol). Aliquots (100 μl) were removed at frequent intervals, placed on 1.2 μM GF/C filters (Whatman International, Maidstone, U.K.) and washed once with 6 ml of 0.85% NaCl poured over the filters under vacuum pressure. Additional aliquots were simultaneously removed for determination of protein content (protein assay kit, Bio-Rad Laboratories, Richmond, Calif.). Radioactivity on the filters was quantified using a Beclkan LS7000 scintillation counter. Results were expressed as nanograins of mitomycin per milligram of cell protein.

[0306] Results

[0307] A gene encoding a transmembrane protein is physically linked to mrd. DNA sequence analysis of a cosmid clone containing the mrd locus, a previously characterized MC resistance determinant (Sheldon et al., 1997), identified an open reading frame (ORF) encoding a polypeptide predicted to be highly hydrophobic that shows similarity to a variety of antibiotic export proteins in drug-producing actinomycetes. Significantly, the gene (mct, SEQ ID NO:72) encoding the putative mitomycin exporter (MC-translocase; MCT) protein is located within 5 kb of mrd (SEQ ID NO:64) and is physically linked to the mitomycin biosynthetic gene cluster (FIG. 15).

[0308] Sequence analysis of the mct locus. Nucleotide sequence analysis of cosmid clone pDHS7547 revealed an ORF predicted to start with the ATG codon at position 132 and end with the TGA codon at nucleotide 1587 (FIG. 16), resulting in a 484 amino acid polypeptide with a predicted molecular weight of 50,023 daltons. Comparison of the deduced amino acid sequence of the mct gene with proteins in the available databases revealed significant similarity to several integral membrane proteins that confer drug resistance. These include the CmcT protein from the cephamycin producer, Nocardia lactamdurans (Coque et al., 1993), the Pur8 protein from the puromycin producer, Streptomyces alboniger (Tercero et al, 1993), the Mmr protein from the methylenomycin producer, Streptomyces coelicolor (Neal and Chater, 1987), and the LmrA protein from the lincomycin producer, Streptomyces lincolnensis (Zhang et al., 1992). The similarities of the mct gene product and related proteins extend over the entire sequences, with the highest levels of similarity found within the amino-terminal regions (FIG. 17).

[0309] Within the N-terminal regions of several antibiotic efflux proteins, including Mmr and LmrA, several highly conserved structural motifs have been noted. The β-turn motif (VxGxLxDxxGRKxxxL), found within the highly conserved cytoplasmic loop sequence separating transmembrane domains two and three of most eukaryotic and prokaryotic transport proteins, is clearly evident in MCT at positions 79-95 (FIG. 16). A motif (LDxTVxNVALP) found at the end of transmembrane domain one, specific to the 14 transmembrane segment family (Paulson and Skurray (1993)) is present in MCT at positions 41-51 (FIG. 16). In addition, several other invariant motifs are apparent in the MCT sequence.

[0310] Transmembrane proteins that mediate resistance to antibiotics and antiseptics by active efflux are highly related, usually containing 12 or 14 transmembrane regions. Notably, the actinomycete drug transport proteins that share homology with MCT appear to contain 14 transmembrane spanning regions and constitute a family of drug resistance translocases. Utilizing the membrane structure and topology program MEMSAT (University College, London), and hydropathy analyses based on the algorithm of Kyte and Doolittle (1982), a prediction of 14 transmembrane spanning domains was made for the deduced amino acid sequence of MCT (FIG. 18).

[0311] Inactivation of mct results in greater sensitivity to MC. To establish a physiological role for MCT in S. lavendulae, the corresponding gene (mct) was inactivated by insertion of the aphII gene from transposon Tn5 to give pDHS7704. After conjugal transfer of pDHS7704 from E. coli to S. lavendulae and growth of the transconjugants under selective conditions, targeted replacement of native mct was achieved by double crossover homologous recombination. Gene disruption was confirmed by Southern blot hybridization of total DNA from the S. lavendulae wild-type and mutant with a DNA probe that included the mct locus. Analytical digests of the genomic DNA resulted in detection of the predicted band shifts in the mutant and wild-type strains (FIG. 19). The S. lavendulae mct disruption mutant strain (MM105) exhibited an approximately 25-fold reduction in resistance to MC when exposed to 100 μg of MC per ml of medium (Table 3). In media lacking MC, the growth kinetics of the strain MM105 was comparable to the wild-type S. lavendulae strain. TABLE 3 Resistance of S. lavendulae strains to varying concentrations of MC Plate count CFU/ml Strain Concentration S. lavendulae mct mutant of MC (μg/ml) S. lavendulae wild-type (MM 105) 10 >10⁷ >10⁷ 20 >10⁷ >10⁷ 40 5.3 × 10³ 2.6 × 10³ 80 2.6 × 10³ 2.4 × 10² 100 2.0 × 10³ 8.0 × 10¹

[0312] Expression of mct in E. coli. To investigate further the function of mct, heterologous expression of the gene in E. coli was pursued. mct was amplified by PCR and cloned into the protein expression vector pET17b to give pDHS7023. pDHS7023 was then introduced into E. coli BL21(DE3) to give strain PJS102. After disruption of the cells by sonication, MCT was found to be associated mainly with the membrane fraction of the cell lysate, as expected for an integral membrane protein. To determine if strain PJS102 was resistant to MC, cultures were grown up and plated on agar medium containing various concentrations of MC. Significantly, IPTG-induced cultures of PJS102 exhibited resistance to MC at drug concentrations 5-fold greater than those for E. coli BL21(DE3) containing vector alone (Table 4). TABLE 4 MC resistance of mct, mrd expressing E. coli strains Plate count CFU/ml Strain Concentration BL21 (DE3):: of MC (μg/ml) pET17b PJS100 PJS102 PJS103 0.0 >10⁷ >10⁷ >10⁷ >10⁷ 0.01 >10⁷ >10⁷ >10⁷ >10⁷ 0.1 7.3 × 10³ >10⁷ >10⁷ >10⁷ 0.5 3.2 × 10² >10⁷ 2.1 × 10⁵ >10⁷ 1.0 0.0 3.3 × 10⁶ 5.9 × 10⁴ >10⁷ 2.5 — NA^(a) 2.0 × 10² >10⁷ 5.0 — 2.7 × 10⁶ 0.0 >10⁷ 10 — 6.1 × 10⁵ — >10⁷ 20 — 2.5 × 10⁵ — >10⁷ 30 — 5.0 × 10² — >10⁷ 60 — 0.0 — >10⁷ 80 — — — 1.4 × 10⁵ 100 — — — 9.6 × 10³ 150 — — — 3.0 × 10¹ Mitomycin B — >10^(7b) NA^(c) >10^(7d)

[0313] Co-expression of mct and mrd in E. coli. To address the notion that MRD and MCT proteins participate as components of a binding protein-dependent drug export system, the mct and mrd genes were co-expressed in E. coli. From plasmid pDHS7006 (mrd expression construct) (Sheldon et al., 1997), a DNA fragment containing the mrd gene under the control of the T7 promoter was ligated into pDHS7023 to give pDHS7024. pDHS7024 was then introduced into E. coli BL21 (DE3) to give strain PJS103. To determine if strain PJS103 was resistant to MC, cultures were grown up and plated on agar medium containing various concentrations of MC. Significantly, IPTG-induced cultures of PJS103 exhibited resistance to MC at drug concentrations 300-fold greater than those for E. coli BL21(DE3) containing vector alone (150 μg/ml vs. 0.5 μg/ml of MC; Table 4). In addition to PJS103 maintaining levels of resistance over that of the vector control strain, co-expression of mct and mrd confers MC resistance at drug concentrations 5 and 60-fold greater compared to PJS100 (containing the mrd gene alone) (Sheldon et al., 1997) or strain PJS102 (containing the mct gene alone), respectively. Strain PJS103 also displayed high-level resistance to mitomycin B (Table 4), a mitomycin also produced by S. lavendulae.

[0314] MC uptake by E. coli cells expressing mct, mrd or mct/mrd. Since the deduced amino acid sequence of the mct gene was similar to antibiotic export proteins, reduced accumulation of MC in MCT-expressing cells would be expected. An assay, modeled after experiments used to study tetracycline efflux-mediated resistance in E. coli (Levy and McMurry, 1978), was designed to study the uptake of [³H]-MC by the susceptible vector control and resistant mct, mrd and mct/mrd expressing E. coli strains.

[0315] MC accumulation by the susceptible vector control strain (BL21(DE3)::pET17b) was found to reach a maximum level at 5 minutes and thereafter maintained at constant concentrations. In contrast, the quantity of MC accumulation in the resistant, mct-expressing strain (PJS102) was only 25% of the susceptible control at 5 minutes, and thereafter remained at reduced concentrations (FIG. 21). Reduced accumulation of drug in PJS102 suggests that mct encodes a protein that facilitates MC export from the cell. To determine if the co-expression of mct and mrd in E. coli also resulted in reduced accumulation of MC, strain PJS103 was analyzed using the [3H]-MC uptake assay. Analyses of drug uptake by cultures of strain PJS100 (Sheldon et al., 1997) were also performed to determine drug accumulation levels in this MC resistant E. coli strain.

[0316] The results show a clear difference in MC accumulation between the MC sensitive and resistant strains. Compared to E. coli cells bearing vector alone, MC accumulation in PJS103 was only 35% at 5 minutes and thereafter remained at reduced concentrations. The accumulation of drug in strain PJS103 was found to parallel that of strain PJS102, albeit at slightly higher levels (about 23% greater) of drug over the course of the experiment. Interestingly, strain PJS100, although resistant to significant concentrations of MC, accumulated drug to levels 42% higher than the drug-sensitive vector control at 30 minutes (FIG. 20).

[0317] Discussion

[0318] Most antibiotics inhibit bacterial growth by binding to proteins or other macromolecular components that involve essential metabolic processes of the cell (Cundliffe, 1992). For instance, DNA alkylation by MC results in disruption of chromosomal replication leading to cell death (Iyer and Szybalski, 1964). In many antibiotic-producing streptomycetes, macromolecular target site(s) are likewise susceptible to endogenous cytotoxic compounds (that is certainly the case in S. lavendulae). Thus, pumping the antibiotic out of the cell at a rate equal to its production and/or re-uptake would prevent drug access to intracellular target sites. Based on the levels of drug found in most antibiotic fermentation broths (concentrations of intracellular drug being low), it is apparent that drug-producing organisms often depend on efficient antibiotic transport mechanisms. Indeed, a growing number of membrane systems implicated in transport (and therefore resistance) of a variety of antibiotics have been discovered in drug-producing streptomycetes (Mendez and Salas, 1998; Paulsen et al., 1996).

[0319] In general, genes coding for drug export proteins are physically linked to the corresponding biosynthetic genes within the genome of the antibiotic-producing microorganism. Presumably, the tight linkage of antibiotic export and biosynthetic genes ensures coordinate gene regulation. Interestingly, the presence of back-to-back and overlapping divergent promoters of antibiotic export and regulatory genes has been observed within the tetracenomycin (Guilfoile and Hutchinson, 1992) and actinorhodin (Caballero et al, 1991) biosynthetic gene clusters. Conforming to this example, S. lavendulae possesses a gene coding for an integral membrane drug export protein within the mitomycin biosynthetic gene cluster. Analysis of the deduced amino acid sequence of MCT revealed several similarities with actinomycete proteins predicted to function as drug exporters. By virtue of homology to tetracycline resistance proteins, which have been shown to use proton motive force to energize transport (Littlejohn et al., 1992), the actinomycete drug resistance translocases cited in this study are predicted to power excretion by a proton-dependent electrochemical gradient. It has been suggested that highly conserved sequences within the amino-terminal regions of these proteins play a role in proton translocation (Rouch et al., 1990), while the less well-conserved C-terminal regions may be involved in drug binding (Paulsen et al., 1996; and references therein) or recognition of a protein-drug complex.

[0320] Disruption of mct in S. lavendulae resulted in a 25-fold increase in sensitivity to exogenously added MC, providing evidence that MCT maintains a role in providing drug resistance in S. lavendulae. Although the effect is significant, alternative mechanisms of cellular self-protection clearly continue to operate. This evidently includes MCRA, the novel redox-relay protein that re-oxidizes activated MC in S. lavendulae. It is also likely that unidentified xenobiotic transporters provide an alternative mode of drug transport in the absence of MCT, albeit with lower efficiency.

[0321] In order to probe the ability of MCT to transport drug in the presence and absence of the MC binding protein, accumulation of [³H]-MC in E. coli was analyzed. Expression of mct in E. coli resulted in MC-resistant cultures that accumulated lower levels of drug than strains bearing vector control (FIG. 20). Interestingly, strain PJS102 (expressing mct only) accumulates less drug intracellularly than strain PJS103 (expressing mrd and mct) (FIG. 20). Increased drug accumulation in strain PJS100 may lend support to the model of equimolar binding between MRD and MC (Sheldon et al., 1997). Significantly higher levels of drug accumulation in strain PJS100 may be the result of intracellular sequestration of MC by MRD. Accordingly, the presence of MRD could also account for the slightly greater levels of MC accumulation in strain PJS103 (co-expressing mct-mrd) as compared to strain PJS102 (expressing mct alone). Comparable to binding protein-dependent import systems (Miller et al., 1983), the binding of MC by MRD may be rate-limiting in the drug excretion process.

[0322] Taken together, these results suggest that cellular protection afforded by MCT is a function of drug transport from the cytoplasm. Interestingly, co-expression of mrd and mct in E. coli led to cultures that are dramatically more resistant to exogenously added drug. While normally required for the transport systems with which they are associated, in many instances binding-proteins are not integral to the process of solute translocation (Higgins et al., 1990). Similarly, the presence of MRD is not required for MC translocation but dramatically enhances drug tolerance. Hence, the binding protein (MRD) may be considered an accessory component, a rather specific adaptation required for optimal drug resistance. The drug-resistance phenotype of E. coli strains expressing mct alone and in combination with mrd along with the MC uptake analysis of these strains provides evidence that MRD and MCT are components of a novel drug transport system. Such a resistance mechanism, sequestering the intact drug for efficient excretion to the environment, represents a unique cellular strategy for self-preservation by the MC-producing organism.

EXAMPLE 4

[0323] Characterization of MitM in the Mitdmycin Biosynthetic Pathway

[0324] Since the identification of the anti-tumor antibiotic mitomycin C from cultures of S. lavendulae in the 1950's (Hata et al., 1956), several dozen new mitomycins with varying methylation and amination patterns on the common mitosane skeleton have been isolated. Biosynthetic studies in the 1970's and 1980's showed that the mitomycins are produced by convergent biosynthesis of the precursors 3-amino-5-hydroxy benzoic acid (Anderson et al., 1980), D-glucosamine (Hornemann et al., 1974), carbamoyl phosphate (Hornemann et al., 1975), and S-adenosyl methionine (Bezanson et al., 1971). However, the specific sequence of the reactions required for assembly of these important compounds remains unknown.

[0325] The varying activity and toxicity of the anti-tumor mitomycin family of compounds is determined by the methoxy and amino substitution patterns present on the mitosane skeleton (Kunz et al., 1991). To study the late mitomycin biosynthetic steps that give rise to the diversity of mitomycins isolated from nature, molecular genetic methods have been employed to understand the role of the methyltransferases in mitomycin biosynthesis. The mitomycin biosynthetic gene cluster has been identified (Mao et al., 1999a; see Examples 1-3) and a conjugative method for DNA transfer and genetic manipulation of the biosynthetic pathway has been developed (Mao et al., 1999b; Examples 1-3). S-adenosyl methionine is known to label the C7, C9a, and the aziridine O and N methyl groups (Bezanson et al., 1971). The methyltransferase reaction has been putatively assigned (Altschul et al., 1990) to the products of the mitM, mitN and mmcR genes by sequence homology to other known secondary metabolite methyltranferases (Kagan et al., 1994; Lacalle et al., 1991; Madduri et al., 1993; and Shi et al., 1996). A detailed understanding of the sequence of reactions carried out by these enzymes is expected to explain the basis of the diversity of the mitomycins produced. Genetic manipulation of the mitomycin producer, bioassay guided isolation and overexpression of the MitM enzyme has allowed the characterization and conversion of an accumulated intermediate from the methyltransferase mitM deleted mutant, S. lavendulae MM107. In addition to understanding the late mitomcyin biosynthetic steps, the manipulation of the mit/mmc pathway can result in the production of novel mitomycins, e.g., through combinatorial biosynthetic manipulation of the pathway. The results which are described below indicate the sequence of late stage methylations that give rise to the diversity of the isolated mitomycins and evidence the production of a novel mitomycin compound.

[0326] Mitomycin C production was absent in the chromosomally in frame mitM gene deleted S. lavendulae mutant MM107 (see Example 5), however, the observation of a small amount of activity against B. subtilis allowed a bioassay guided isolation of a novel compound, 9a-demethoxy mitomycin A, to take place. The isolation method consisted of applying the crude ethyl acetate extract of the culture broth culture to a Sephadex LH-20 column followed by preparative and analytical reverse HPLC to provide 9a-demethoxy mitomycin A (1) (FIG. 27A) at a yield of 10-30 μg per liter of culture.

[0327] Observation of the signals for protons H1, H2, H3α, H3β, Me6, H9, H10, and H10′ in the ¹H NMR of the new compound indicated the mitomycin skeleton was present (Data for 9a demethoxy mitomycin A: ¹H NMR (CD₃CN, 800 MHz). 1.77, (s, C6-methyl), 2.74 (m, H2), 2.81 (d, J=m, H1), 3.31 (dd, J=4.3, 11.3 Hz, H9), 3.39 (d, J=12.8 Hz, H3α), 3.84 (, J=12.8, H3β), 3.95 (s, C7-OCH₃), 4.16 (dd, J=10.4, 11.3 H10′), 4.71 (dd, J=4.3, 10.4, H10); ¹H NMR data for 9-epi mitomycin B (2): (CD₃CN, 800 MHz). 1.78, (s, C6-methyl), 2.22 (s, N-aziridine Me), 2.31 (m, overlapped H1, H2), 3.31 (ddJ=4.9, 9.8 Hz, H9), 3.38 (d, J=12.2 Hz, H3α), 3.84 (d, J=12.2, H3β), 3.96 (s, C7-OMe), 4.06 (dd, J=9.8, 12.2 Hz, H10′), 4.67 (dd, J=4.9, 12.2 Hz, H10)). The presence of the methoxy singlet at 4.0 ppm and the absence of other methoxy or aziridine-N methyl signals at 3.3 ppm and 2.2 ppm, respectively, led to a preliminary structure consistent with 9a-demethoxy mitomycin A. A m/z of 374.0074 (1.9 ppm of calc'd) from HRESIMS of [M+K]⁺ confirmed the formula for this structure. The ¹³C NMR signals for the carbon skeleton was observed by HMQC and HMBC experiments due to difficulties in obtaining sufficient signal from directly detected ¹³C NMR experiments (¹³C NMR (CD₃CN, 800 MHz, inversely detected by HMBC and HMQC, ppm, atom). 37.4, C1 , 33.4 m C2, 49.6, C3, 152.0, C4, 184, C5, 7.5, C6a (methyl), 125, C6, 160.4, C7, 114.2, 62.0, C7a (O-methyl), C8a, 103, C9a. 49.4, C9, 157.9, C10a (carbamoyl carbonyl), 63.0, C10. C8 was not detected by HMBC). In addition to providing the ¹³C NMR spectrum, the heteronuclear correlation experiments demonstrated the presence of the quinone moiety, the connectivities for the 7-methoxy and C6 methyl groups to the aminoquinone group, and the presence of the carbamoyl carbon from a H10 to C10a correlation.

[0328] Determination of the stereochemistry for the C9-C10 bond was first attempted by difference nOe, NOESY and ROESY experiments, however, the lack of a clear nOe effect between the protons on C10 and the H1, H2 and H3 protons prevented assignment of stereochemistry. Overexpressed MitM from E. coli (Example 5) was used to convert 9a-demethoxy mitomycin A to an N-aziridine methylated product. The 2.2 ppm methyl signal and the upfield shift of the H1 and H2 signals from 2.90 and 2.85 to merge at 2.5 ppm are consistent with an N-aziridine methyl group in the mitomycin series of compounds. By thin layer chromatography (silica gel, 9:1 CHCl₃:CH₃OH) it was apparent the product (R_(f)=0.21) was not mitomycin B (R_(f)=0.26), and thus must be 9-epi mitomycin B with C9-C10 in the β configuration. The stereochemistry of 1 and 2 at the C9a, C1 and C2 carbons were assigned based on biosynthetic consistency with previously isolated mitomycins (Hornemann et al., 1985).

[0329] The activity of MitM upon 9a-demethoxy mitomycin A was examined to identify the role of this enzyme in the mitomycin C biosynthetic pathway. MitM was found to have a pH optimum of 8 (above a certain pH the SAM substrate decomposes, while below pH 7 the mitomycins are known to be unstable). As described above, MitM was shown to methylate specifically the aziridine nitrogen of 9a-demethoxy mitomycin A while leaving the 9a oxygen untouched. Addition of a methyl group was confirmed by the HRESIMS of 2 which provided a m/z=[M+Na]⁺ 372.1186 (3.9 ppm of calc'd). Kinetic experiments with varying amounts of 9a-demethoxy mitomycin A at 500 μM SAM allowed determination of a Km of 263 μM and a k_(cat) of 0.11 s⁻¹. For SAM the Km of 30 μM, and a k_(cat) 23 μM were determined, however, these experiments took place with 400 μM mitomycin A due to limited amounts of 9a-demethoxy mitomycin A. Using the same conditions that provided activity for mitomycin A and 9a-demethoxy mitomycin A enzymatic conversions, no conversion products were observed for mitomycins B, C, D, and G.

[0330] Discussion

[0331] The identification and sequencing of the mitomycin C biosynthetic pathway allowed the assignment of methyltransferase functions to the proteins encoded by the genes mitM, mitN, and mmcR (Mao et al., 1999a). A conjugative transfer method for S. lavendulae was used to create mutants containing either deleted or insertionally disrupted versions of these genes on the S. lavendulae chromosome (Mao et al., 1999b). Examination of the mitM deleted mutant, MM107, for accumulated intermediates led to the isolation and characterization of the new mitomycin, 9a-demethoxy mitomycin A. The presence of a demethylated mitomycin was expected from the previous assignment of methyltransferase activity to mitM based on homology to other secondary and primary metabolite methyltransferases. The identification of this novel compound reaffirms that knockout mutants in the mitomycin C biosynthetic pathway are an effective strategy to determine the biosynthetic sequence of this important anti-cancer agent.

[0332] The isolated intermediate 9a-demethoxy mitomycin A can potentially be methylated at either the 9a oxygen or the aziridine nitrogen by SAM. Reacting 9a-demethoxy mitomycin A with MitM provided 9-epi mitomycin B, indicating that this methyltransferase specifically acts upon the aziridine nitrogen. A further probe of MitM activity was carried out with the mitomycins A, B, C, D and G. Only mitomycin A was methylated at the aziridine nitrogen by MitM to produce mitomycin F. While mitomycin C also has a free aziridine nitrogen that can potentially be methylated to form porfiromycin this was not observed in the reaction with MitM. Thus, the reactivity of MitM appears to be general with regard to the aziridine position of the mitomycins with a C7-methoxy group such as mitomycin A and 9a-demethoxy mitomycin A, but the presence of the C7-amine of mitomycin C prevents this activity. Due to lack of a suitable substrate in the mitomycin B series of compounds (α C9-10 bond configuration, unmethylated aziridine) the specificity of MitM with regards to this stereochemistry could not be determined.

[0333] It has been observed that mitomycin A titers first rise and then fall as mitomycin C titers increase (Hornemann, 1981). This implied that mitomycin C is produced from mitomycin A. To date there has no been direct biochemical evidence for this transformation. From the results described herein, it was found that affecting the enzyme that converts mitomycin A to F and 9a-demethoxy mitomycin A to 9-epi mitomycin B led to the abolition of mitomycin C production. When the late methylations for the mitomycins with C9 α stereochemistry are examined, scheme 1 (FIG. 28), an early split from a putative demethylated precursor Unknown A can be hypothesized. This putative precursor can be methylated at the C7 position to form 9a-demethoxy mitomycin A and enter into the mitomycin A series of compounds, or an amine may added at the C7 position to form Unknown B and start the mitomycin C group of compounds. Whether the split occurs early as shown in scheme 1, or if there is an aminotransferase that acts upon the either mitomycin A or one of its related C7 methoxy compounds, is not known.

[0334] Thus, it appears that MitM is responsible for methylating the aziridine nitrogen to convert 1 to 2 while also converting mitomycin A to F. Hence, the molecular genetic manipulation of the mitomycin biosynthetic pathway has provided information on the sequence of the reactions involved in mitomycin C biosynthesis. Compound 1 has not been previously synthesized or isolated (Yoda et al., 1993) and 2 is only known from synthesis using mitomycin B as the starting material (Kasai et al., 1989).

EXAMPLE 5 Genetic and Enzymnatic Analysis of Two Aziridine N-methyltransferases (MitMn) in the Mitomycin Biosynthetic Gene Cluster

[0335] Mitomycin C (MC) is a clinically important anti-tumor antibiotic and has been widely used for combination cancer chemo- and radio-therapy for more than three decades (Henderson, 1993). In an effort to select more potent and low toxicity anti-tumor drugs, chemists and microbiologists have synthesized various mitomycin analogs (Kasai et al., 1995). The first isolated mitomycin antibiotics were mitomycin A (MA) and mitomycin B (MB) in 1956 in the fermentation of S. lavendulae (Hata et al., 1956). In the following year, Wakaki et al. (1958) found mitomycin C (MC) in the same culture broth when it did not produce MA and MB. The mitomycin family was further enriched by identification of a group of minor mitomycin components from the fermentation broth in the ensuing years (Shirahata et al., 1981). These compounds had a common heterocyclic ring system: the mitosane core, while they had different modifications (e.g., methylation, amination, or both) at N-1a, C-7 and C-9 positions (FIG. 27B). The methyl groups at these positions had been demonstrated to be derived from L-methionine since radio-labeled L-[¹⁴CH₃] methionine can specifically label the O- and N-methyl groups in MA while antibiotic production was inhibited by the methionine antagonist D,L-ethionine (Kirsch et al., 1964; Bezanson et al., 1971). Another experiment in the early 1970s had shown that one of the demethylated derivative of MC can be converted to MA by the cell-free extracts of S. lavendulae when supplied with exogenous L-methionine, ATP and Mg2+, while at the same condition, the C10 decarbamoylated form can not (Nishikohri et al., 1975). These experiments not only demonstrated the existence and involvement of methyltransferases in MC biosynthesis, but also indicated those enzymes had some degree of substrate specificity.

[0336] The final step for methyl transfer utilizing L-methionine as the methyl donor is usually catalyzed with a group of enzymes called S-adenosylmethionine (SAM)-dependent methyltransferases. In the presence of ATP, Mg2+, L-methionine was first converted by synthetase to SAM. These methyltransferases then transfer the methyl group from SAM to either macromolecules such as proteins, nucleic acids (RNA or DNA), phospholipids, polysaccharides, or small molecules such as drugs, hormones, and neurotransmitters with the formation of methyl ester, methyl ether, methyl thioether, methyl amine, methyl amide, and other derivatives (Weinshilboum et al., 1999). Though there existed three loosely conserved SAM or S-adenosyl-homocysteine binding motifs within the enzymes, methyltransferases do not display the same degree of sequence homology as had been fond among the cytochromes P450, and no similar overarching classification system had been developed (Kagan et al., 1994). The most commonly found small molecule methyltransferases were O-methyltransferase which has been widely found in both eukaryotes (such as catechol O-methyltransferase; see Lundstrom et al., 1995) and prokaryotes. Most of the reported macrolide antibiotic biosynthetic methyltransferases were revealed to be in this category. These genes includes: eryG for erythromycin (Haydock et al., 1991), tcmN for tetracenomycin (Summers et al., 1992), dnrK for carminomycin (Madduri et al., 1993), dauK for daunomycin (Dickens et al., 1995), orf14 for rifamycin (August et al., 1998), aveD for avermectin (Ikeda et al., 1999),fkbM for FK506 (Motarnedi et al., 1996), mdmC for midecamycin (Hara et al., 1992), and dmpM for puromycin (Lacalle et al., 1991). Small molecule SAM dependent N-methyltransferase is quite common in mammalian cells in the metabolism of many endogenous neurotransmitters and hormones, e.g., phenylethanolamine N-methyltransferase (Vance et al., 1998), histamine N-methyltransferase (Okinga et al., 1995), and glycine N-methyltransferase (Ogawa et al., 1998). Though N-methyltransferase genes were also proposed in antibiotic biosynthetic gene clusters (pur5 in puromycin gene cluster, see Tercero et al., 1996), spcM for spectinomycin biosynthesis (AAD28488), tylM1 in tylosin cluster (Gandecha et al., 1997), none of their functions has been confirmed

[0337] Three SAM dependent methyltransferases (MmcR, MitM and MitN) were assigned in the MC biosynthetic gene cluster because of the three conserved SAM binding motifs within their protein sequences (Mao et al., 1999). Database analysis revealed that MmcR showed strong sequence similarity with a group of antibiotic O-methyltransferases, whereas both MitM and MitN are most closely related to a group of plant δ-(24)-sterol C-methyltransferases as well as some antibiotic biosynthetic O-methyltransferases (Mao et al., 1999). Considering that both the O- and N-methyl groups in mitomycin molecules were derived from L-methionine and there were three SAM dependent methyltransferases available in the MC cluster, localization of the detailed functions of these methyltransferases in MC biosynthesis will provide insight into determining the details of MC biosynthesis and the substrate specificity of these enzymes.

[0338] Material and Methods

[0339] Bacterial Strains and Cloning Vectors

[0340]E. coli DH5α (Table 5) was routinely used as a cloning host, and propagated on LB media E. coli S17-1 was used as a conjugation host for gene transfer from E. coli to S. lavendulae. Conjugation was performed on AS1 media and double crossovers were selected on R5 media as described previously (Mao et al., 1999). E. coli BL21 (DE3) was the protein overexpression host in NZYM media. TABLE 5 Bacterial strains and plasmids. Strains or Plasmids Relevant Characteristics^(a) Reference E. coli strains DH5α F recA φ80 dlacZ ΔM15 Gibco BRL S17-1 Contains RP4 integrated into the Mazodier chromosome et al., 1989 BL21 (DE3) F ompT hsdS gal dcm Novagen S. lavendulae (DE3) NRRL 2564 Wild type MC producer ATCC MM106 mitM insertional disruption mutant of NRRL This Study 2564 MM107 mitM in frame deletion mutant of This Study NRRL 2564 MM108 mitN in frame deletion mutant of This Study NRRL 2564 Plasmids pET28b Kan^(R) , Protein expression vector Novagen pUC119 Ap^(R) lacZαMCS, E. coli cloning vector Yanisch- Peron et al., 1985 pKC1139 AM^(R) lacZαMCS oriT Bierman rep^(ts) et al., 1992 pDHS7608 mitMN containing 2.5 kb BamH1 This study fragment in pUC119 pDHS7705 mitM disruption construct in pUC119 from This study pDHS7608 pDHS7706 mitM disruption vector in pKC1139 from This study pDHS7705 pDHS7707 mitM deletion construct in pUC119 This study pDHS7708 mitM deletion vector in pKC1139 from This study pDHS7707 pDHS7709 mitN deletion construct in pUC119 This study pDHS7710 mitN deletion vector in pKC1139 from This study pDHS7710 pDHS7801 MitM overexpression vector This study pDHS7802 MitN overexpression vector This study

[0341] pUC119 was the cloning vector in E. coli. pKC1139, an E. coli-Streptomyces conjugative shuttle vector was used to create disruption mutants in S. lavendulae. Plasmid pET28(b) was used for protein overexpression in E. coli BL21 (DE3).

[0342] DNA Manipulation

[0343] The mitM insertional disruption vector pDHS7706 was constructed as follows: the mitMN genes-containing plasmid pDHS7608 was digested with Bpu11021, blunt-ended and then ligated with the 1.4 kb ApaL1-HindIII (aphII) fragment from pFD666 to create pDHS7705. The insertion of the aphII gene in Bpu11021 results in the disruption of mitM. The 3.9 kb BamHI insert from pDHS7705 was then moved to the shuttle vector pKC1139 to generate pDHS7706.

[0344] Three pairs of primers were used to construct the mitM and mitN in-frame deletion vectors pDHS7708 and pDHS7710. For the mitM in-frame deletion vector pDHS7708, primer f1: 5′-GC TCT AGA TCT ACG TCT CCC GCG-3′ (XbaI; SEQ ID NO:146) and primer r1: 5′-GC CTC GAG CAT GGA CGA TCC CTC TCG-3′ (XhoI; SEQ ID NO:147) were used to generate a 1.1 kb mitM upstream fragment (MTF11) by introducing an additional XhoI site right after the mitM translational start codon. A 2.1 kb mitM downstream fragment (MTF23) was amplified with primer f2: 5′-GC CTC GAG CCG GGA AAG TGA GCG GCA-3′ (XhoI; SEQ ID NO:148) and primer r3: 5′-GC AAG CTT CGG CAT CAC GCG CCA-3′ (HindIII; SEQ ID NO:149) by introducing a XhoI site just upstream of the mitM translational stop codon. MTF11 and MTF23 were then digested with XbaI/XhoI and XhoI/HindIII, respectively, and ligated to the XbaI/HindIII digested pUC119 to create pDHS7707. In this case, mitN was left intact, while the whole ORF of mitM was replaced by a XhoI site. The 3.2 kb insert from pDHS7707 was then subcloned into the XbaI/HindIII site of pKC1139 to generate pDHS7708. Similarly, a 1.9 kb mitN upstream fragment (MTF12) and a 1.3 kb downstream fragment (MTF33) were amplified with two pairs of primers: primer f1 and primer r2: 5′-GC CTC GAG CGT CAT GCC OGCT CAC TTT-3′ (XhoI; SEQ ID NO:150) for MTF12 and primer f3: 5′-GC CTC GAG TAG GGC TCC CAC GGG AAG-3′ (XhoI; SEQ ID NO:151) and primer r3 for MTF33. Three way ligation of the XbaI/XhoI digested MTF12, XhoI/HindIII digested MTF33 and the XbaI/HindIII digested pUC119 resulted in pDHS7709, in which the mitN ORF was replaced with a XhoI site while mitM was left intact. pDHS7710 was created by subcloning the 3.2 kb XbaI/HindIII insert from pDHS7709 into pKC1139.

[0345] For the construction of the MitM and MitN overexpression vector, an NdeI site was introduced at the translational start codon of mitM with primer f4: 5′-GGG ATC GCA TAT GCC GCA CTC CGA GCT GTC-3′ (NdeI; SEQ ID NO:152), and mitN with primer f5: 5′-GTG AGC CAT ATG ACG GAA ACC GCG TCC GC-3′ (NdeI; SEQ ID NO:153). With pDHS7608 as template, and M13 /pUC reverse sequencing primer as the other PCR primer pair, a 2.31 kb fragment (for MitM) and a 1.45 kb fragment (for MitN) were amplified, respectively. The resulting fragments were then digested with NdeI/HindIII and subcloned into the corresponding sites of pET28b to generate pDHS7801 (for MitM) and pDHS7802 (for MitN). In both cases, the overexpressed proteins were engineered to include an N-terminal 6×His tag.

[0346] Creation of Disruption Mutant Strains of S. lavendulae

[0347] The mitM insertional disruption mutant S. lavendulae MM106 was created by conjugating the mitM disruption vector pDHS7706 into wild type S. lavendulae NRRL 2564 following the protocol described in Mao et al. (1999). An apramycin, kanamycin double resistant conjugant was selected and propagated on R5 plates at 39° C. for several generations to select for the double crossover mutants. Kanamycin resistant and apramycin sensitive colonies were checked by Southern hybridization to have the correct double-crossover genotype.

[0348] A two-step recombination strategy was used to create the mitM and mitN in-frame deletion mutants MM107 and MM108. In these experiments, pDHS7708 and pDHS7710 were conjugated into MM106, the mitM insertional disruption mutant. Following by the selection of kanamycin and apramycin sensitive colonies from the corresponding double antibiotic resistant conjugants, mitM and mitN were successfully deleted from the chromosome. The deletion of mitM in MM107 and mitN in MM108 were confirmed by Southern hybridization.

[0349] Overexpression and Purification of MitM and MitN

[0350] pDHS7801 and pDHS7802 were transforned into E. coli BL21 (DE3) strain for the overexpression of MitM and MitN, respectively. E. coli cells were grown at 37° C. in NZCM broth containing 50 μg/ml kanamycin until OD₆₀₀=0.6. Expression of MitM (or MitN) was induced by adding IPTG to a final concentration of 0.1 mM and reducing the temperature to 25° C. After incubating the culture overnight, cells were harvested, suspended in ice-cooled lysis buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 10 mM imidazole), and lysed by sonication (6×10 seconds with 10 second pauses at 200-300 W). The supernatant collected by centrifuging the lysate at 10,000 g at 4° C. was used to pass through a Qiagen Ni-NTA column as specified by the manufacturer. The column was eluted twice with wash buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 20 mM imidazole) followed by dissolving the protein in elution buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 250 mM imidazole). The protein eluted from the column was then precipitated with 60% ammonia sulfate, desalted by passing through a Pharmacia PD-10 column, and dissolved in 50 mM pH7.4 potassium buffer with 1 mM PMSF. The protein concentration was determined using the Bradford Assay with BSA as the standard. The final concentration of MitM is 0.38 mg/ml and MitN is 0.71 mg/ml.

[0351] Enzyme assays

[0352] Enzymatic conversion of MitM (and MitN) with different substrates was carried out in a total volume of 100 μl in 50 mM potassium phosphate buffer (pH 7.4), containing 100 μM S-adenosyl-methionine (SAM), an appropriate quantity of substrate, and 5 μg of purified enzyme. The reactions were incubated at 30° C. for 30 minutes and were terminated by twice equal volume of ethyl acetate extraction. The resulting organic extracts were combined, vacuum dried and resuspended in 20 μl methanol for further TLC or HPLC analysis (Mao et al., 1999).

[0353] Product Identification

[0354] Both MitM and MitN can convert MA to a new product, with the TLC and HPLC behaviors identical to MF. For further confirmation, the products isolated and purified from several standard enzyme reactions by HPLC were analyzed by ¹H NMR.

[0355] Kinetic Measurement

[0356] The kinetic data for the conversion of MA to MF by MitM and MitN were obtained by detecting the ME production in 313 nm with HPLC under conditions of initial velocity (5-20 minutes) with 1.9 μg MitM or 1.4 μg MitN. Kinetic parameters (V_(max) and Km) were determined for both substrate MA and SAM from the Lineweaver-Burk double reciprocal plot. For MA, reactions were carried out at a fixed amount of SAM (200 μM) with variable amount of substrate MA (30-300 μM), while the parameters for variable SAM (40-400 μM) were measured at the fixed concentration of MA (200 μM). The calculations were carried out using Kcat computer program by fitting the data to a Michaelis-Menton equation by linear regression.

[0357] Results

[0358] Creation of mitM and mitN Deletional Mutants

[0359] The chromosomal copy of mitM gene was disrupted by inserting a neomycin resistance marker in the middle of its open reading frame. The resultant mutant strain, MM106, abolished MC production. However, unlike the early MC biosynthetic gene (mitA) knockout mutant which totally blocked mitomycin compounds production, MM106 still produced some colorful bioactive mitomycin intermediates (data not shown). Since mitM and mitN are the last two genes within a 14-gene cluster which had the same transcription direction (Mao et al., 1999), insertional disruption of mitM probably affects the downstream mitN gene expression. To avoid such polar effect, a mitM in-frame deletion mutant MM107 was created from MM106 followed by a two step recombination in which the whole mitM ORF was replaced with two amino acids (FIG. 38). Similarly, a mitN in-frame deletion mutant, MM108, was also created from MM106 with the same strategy. Both mutants were selected based on the phenotype changing from kanamycin, apramycin double resistant to double sensitive, and confirmed by the correct band shifts with Southern hybridization (data not shown).

[0360] As shown in FIG. 39, the mitM deletion mutant (MM107) does not produce MC, while the mitN clean deletion mutant (MM108) slightly increases MC production. These results indicate that MitM is a key enzyme in MC biosynthesis, while MitN probably serves in the production of shunt metabolites.

[0361] Overexpression and Purification of MitM and MitN in E. coli

[0362] MitM is a hydrophilic protein (deduced pI=4.66) which comprises of 283 amino acids with a calculated molecular weight (MW) of 31 kD. Similarly, MitN consists of 275 amino acids with a deduced MW of 30 kD and a deduced pI of 4.59. Both MitM and MitN were overexpressed in E. coli using the pT7 promoter. Overexpression of both proteins was achieved by adding very low concentrations of IPTG (0.1 mM), and purification to homogeneity was achieved using a Ni-NTA column (FIG. 40).

[0363] Conversion of MA to MF by Both MitM and MitN

[0364] In vitro assay can not detect any MC conversion from the broth extract of MM107 by either MitM or MitN (data not shown), indicating that MitM is not the last step enzyme in MC biosynthesis. MA, MB, MC, MD and MH were employed as candidate substrates for both MitM and MitN. In the presence of SAM, a similar new product was formed with MA as substrate for both enzymes, while no conversions were detected with other mitomycins. The TLC and HPLC profiles of these new products were exactly the same as those of MF. To further locate the methyl group in the new compounds, the products were purified through HPLC and subjected to NMR analysis. In the ¹H NMR map, standard MA have three peaks for the three corresponding methyl groups: 1.83 (C6a methyl), 3.22 (C9a O-methyl), and 3.99 (C7a O-methyl). The extra N1a methyl group in MF adds a new peak at 2.74, as well as shifts the C9a O-methyl peak to 3.21. Both the MitM and MitN conversion products had the same four peaks (1.83, 2.74, 3.21, 3.99) as appeared in MF. The NMR result furier confirmed that the methyl group was added to N1a position of MA in both conversion products, and both MitM and MitN were N-methyltransferases.

[0365] Kinetic Data of the Conversions

[0366] In a range of pH 5.0-8.0, MitM has an optimal pH at 8.0, and the optimal pH for MitN is 7.4. Kinetic data were determined in pH 7.4 for both enzymes. As shown in Table 6, MitN had a higher (Km=94.7 μM) affinity to MA than MitM (Km=169.2 μM), while MitM had a higher (Km=22.8 μM) affinity to SAM than MitN (86.6 μM). However, MitN had a bigger Vmax value than MitM for either MA or SAM. TABLE 6 Molecular Weight (kD) 31 30 Optimal pH 8.0 7.4 Km for MA [μM] 169.2 +/−4.3 94.7 +/−4.5 Vmax for MA (nmol . mg⁻¹ · min⁻¹)  57.4 +/−4.2 75.9 +/−2.4 Km for SAM [μM]  22.8 +/−2.4 86.6 +/−4.5 Vmax for SAM (nmol . mg⁻¹ · min⁻¹)  29.8 +/−4.5 49.4 +/−5.3

[0367] Discussion

[0368] The discovery that both MitM and MitN are aziridine N-methyltransferases is unexpected. Based on the facts that disruption of mitM blocks MC production, while the mutant broth extract can not be converted into MC by MitM (data not shown), it can be concluded that MitM is a key but not the last step enzyme in MC biosynthesis. Taken together with the mitN disruption result, the functions of MitM and MitN in MC biosynthesis can be pictured as following:

[0369] where A and B are the real substrate and, product of MitM, respectively. C is the product of MitN while the real substrate of MitN can be either the MC biosynthetic intermediates (branching) or downstream mitomycins after MC (tailoring).

[0370] As revealed by protein sequence analysis and in vitro conversion experiments, MitM is an N-methyltransferase. However, since there is no N-methyl group in MC molecule, the function of MitM in MC biosynthesis is questionable. One possibility is that the aziridine N-methylation serves as a temporary protection for other reactions and the methyl group will leave in the end. Another possibility is that in addition to its N-methyltransferase activity, MitM also has other functions, such as the C9a or C7 O-methyltransferase activities (even though it has no reaction with MB, MD and MH).

[0371] Considering the late stage mitomycin modifications, there are two possible routes concerning the order of transamination and methylation at C7 (FIG. 42). Hornemann (1981) had proposed that MA is the natural precursor of MC based on the following observations: 1) S. lavendulae which produces mainly MA and MB in some cultural conditions, produces MC exclusively under other conditions, indicating a close biosynthetic relationship between MA and MC (Wakaki et al., 1958); 2) MC can be obtained from MA simply by treatment of the latter with ammonia; and 3) a similar kind of transamination (from MA to MC) was detected in a feeding experiment of converting radio-labeled MF to porfiromycin (Hornemann, 1981).

[0372] Supporting this view, Tomohiro et al (1983) selected a S. lavendulae mutant strain which abolishes MC production but still accumulates MA and MB.

[0373] MM107 (mitM) blocks MA production while MM108 (mitN) may still produce MA (MA production was checked in both mutants. On TLC plates, the mitM mutant MM107 still produces three visible pink mitomycin compounds with one Rf value less than MC and the other two less than MA (FIG. 43)).

[0374] Both MitM and MitN have been confirmed to methylate MA but not MC, which indicates a certain level of substrate specificity. However, disruption and in vitro conversion results indicate that MA is probably not the real natural substrate for both MitM and MitN. Kinetic data revealed that both MitM and MitN had lower affinity to substrate but comparable affinity to SAM comparing to the purified Streptomyces antibiotic biosynthetic O-methyltransferases. These characterized enzymes include: macrocin O-methyltransferase (MOMT) and demethylmacrocin O-methyltransferase (DMOMT) for tylosin biosynthesis in S. fradiae (Bauer et al., 1988; Kreuzman et al., 1988), carminomycin O-methyltransferase (CMT) for daunomycin biosynthesis in S. sp. Strain C5 (Connors et al., 1993), 31-O-desmethylimmunomycin O-methyltransferase (DIMT) for immunomycin biosynthesis in S. hygroscopicus var ascomyceticus (Byrne et al., 1993), and 31-O-demethylFK506 O-methyltransferase (FKMT) for FK506 biosynthesis in S. sp. MA6858 (Shafiee et al., 1994). The Km values of MitM (169 μM) and MitN (95 μM) for MA are much higher than that of MOMT (5 μM), DMOMT (6 μM), CMT (0.5 μM), DIMT (11 μM), and FKMT (23 μM) for their corresponding substrates. For SAM, the Km of MitM (23 μM) is very close to the MOMT (23 μM), CMT (25 μM), DIMT (13 μM), and FKMT (28 μM) while the Km of MitN (87 μM) is comparable to DMOMT (111 μM).

[0375] The data described herein supports the hypothesis that both MitM and MitN are SAM-dependent methyltransferases for mitomycin biosynthesis. Nevertheless, the outcome that both of them are aziridine N-methyltransferases is unexpected. It is possible that MA, the hypothesized precursor for other mitomycins, is a favorable substrate for many a modification enzyme. Different expression level and kinetic balancing of such enzymes will in the end determine the diversity of mitomycin production. Thus, the results not only demonstrate aziridine N-methyltransferase activity but evidence the ability to engineer the biosynthesis of other mitomycin analogs.

[0376] In summary, two putative SAM dependent methyltransferases (mitM and mitN) previously assigned in the MC biosynthetic gene cluster were deleted in the MC producer S. lavendulae NRRL 2564. While the mitN deletion mutant MM108 slightly increases MC production, the mitM deletion mutant MM107 produces no MC but accumulates other bioactive intermediates. The disruption result indicates that MitM is a key enzyme for MC biosynthesis, whereas MitN probably serves as a branching or tailoring enzyme that leads to other mitomycins. Both MitM and MitN were overproduced in E. coli and purified to homogeneity. In the presence of SAM, both MitM and MitN can convert mitomycin A (MA) to mitomycin F (MF), but have no reactions with MB, MC, MD and MH. Kinetic analysis revealed that MitM had a higher affinity to SAM but lower affmity to MA than MitN. Thus, both MitM and MitN are novel mitomycin aziridine N-methyltransferases.

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[0559] While the present invention has been described in connection with the preferred embodiment thereof, it will be understood many modifications will be readily apparent to those skilled in the art, and this application is intended to cover any adaptations or variations thereof. It is manifestly intended this invention be limited only by the claims and equivalents thereof.

1 153 1 115 PRT Streptomyces lavendulae 1 Arg Ile Gly Ala Gly Ser Arg Val Leu Asp Leu Gly Cys Gly Val Gly 1 5 10 15 Thr Pro Gly Val Arg Ile Ala Arg Leu Ser Gly Ala His Val Thr Gly 20 25 30 Ile Ser Val Ser His Glu Gln Val Val Arg Ala Asn Ala Leu Ala Glu 35 40 45 Glu Ala Gly Leu Ala Asp Arg Ala Arg Phe Gln Arg Ala Asp Ala Met 50 55 60 Asp Leu Pro Phe Glu Asp Glu Ser Phe Asp Ala Val Ile Ala Leu Glu 65 70 75 80 Ser Ile Ile His Met Pro Asp Arg Ala Gln Val Leu Ala Gln Val Gly 85 90 95 Arg Val Leu Arg Pro Gly Gly Arg Leu Val Leu Thr Asp Phe Phe Glu 100 105 110 Arg Ala Pro 115 2 114 PRT Streptomyces lavendulae 2 Arg Leu Ala Pro Gly Glu Arg Val Leu Asp Val Gly Ser Gly Asn Gly 1 5 10 15 Lys Ala Thr Leu Arg Ile Ala Ala Arg His Gly Val Arg Ala Thr Gly 20 25 30 Val Ser Ile Asn Pro Tyr Gln Val Gly Leu Ser Arg Gln Leu Ala Glu 35 40 45 Lys Glu Gly Asp Glu Ala Thr Glu Phe Arg Ile Gly Asp Met Leu Ala 50 55 60 Leu Pro Phe Pro Asp Gly Ser Phe Asp Ala Cys Tyr Ala Ile Glu Ser 65 70 75 80 Ile Cys His Ala Leu Glu Arg Ala Asp Val Phe Thr Glu Ile Ala Arg 85 90 95 Val Leu Arg Pro Gly Gly Arg Val Thr Val Thr Asp Phe Val Leu Arg 100 105 110 Arg Pro 3 115 PRT Streptomyces lavendulae 3 Asp Phe Ser Gly Ala Ala Thr Ala Val Asp Ile Gly Gly Gly Arg Gly 1 5 10 15 Ser Leu Met Ala Ala Val Leu Asp Ala Phe Pro Gly Leu Arg Gly Thr 20 25 30 Leu Leu Glu Arg Pro Pro Val Ala Glu Glu Ala Arg Glu Leu Leu Thr 35 40 45 Gly Arg Gly Leu Ala Asp Arg Cys Glu Ile Leu Pro Gly Asp Phe Phe 50 55 60 Glu Thr Ile Pro Asp Gly Ala Asp Val Tyr Leu Ile Lys His Val Leu 65 70 75 80 His Asp Trp Asp Asp Asp Asp Val Val Arg Ile Leu Arg Arg Ile Ala 85 90 95 Thr Ala Met Lys Pro Asp Ser Arg Leu Leu Val Ile Asp Asn Leu Ile 100 105 110 Asp Glu Arg 115 4 115 PRT Streptomyces anulatus 4 Asp Phe Ser Ser Tyr Gly Thr Val Val Asp Ile Gly Gly Ala Asp Gly 1 5 10 15 Ser Leu Leu Ala Ala Val Leu Ser Ala His Pro Gly Val Glu Gly Val 20 25 30 Val Phe Asp Ser Pro Glu Gly Ala Arg Asp Ala Ala Ala Thr Leu Asp 35 40 45 Ala Ala Gly Val Gly Glu Arg Gly Arg Val Glu Thr Gly Asp Phe Phe 50 55 60 Thr Arg Val Pro Gly Gly Gly Asp Leu Tyr Val Leu Lys Ser Ile Leu 65 70 75 80 His Asp Trp Ser Asp Ala Arg Ser Ala Asp Ile Leu Arg Thr Val Arg 85 90 95 Ala Ala Met Pro Ala His Ala Arg Leu Leu Val Val Glu Val Leu Leu 100 105 110 Pro Asp Thr 115 5 117 PRT Streptomyces glaucescens 5 Gly Met Glu Arg Phe Ser Arg Ile Ala Asp Leu Gly Gly Gly Asp Gly 1 5 10 15 Trp Phe Leu Ala Gln Ile Leu Arg Arg His Pro His Ala Thr Gly Leu 20 25 30 Leu Met Asp Leu Pro Arg Val Ala Ala Ser Ala Gly Pro Val Leu Glu 35 40 45 Glu Ala Lys Val Ala Asp Arg Val Thr Val Leu Pro Gly Asp Phe Phe 50 55 60 Thr Asp Pro Val Pro Thr Gly Tyr Asp Ala Tyr Leu Phe Lys Gly Val 65 70 75 80 Leu His Asn Trp Ser Asp Glu Arg Ala Val Thr Val Leu Arg Arg Val 85 90 95 Arg Glu Ala Ile Gly Asp Asp Asp Ala Arg Leu Leu Ile Phe Asp Gln 100 105 110 Val Met Ala Pro Glu 115 6 115 PRT Amycolatopsis mediterranei 6 Pro Leu Arg Ala Gly Asp Arg Leu Leu Asp Ile Gly Cys Gly Asn Gly 1 5 10 15 Glu Pro Ala Ile Arg Met Ala Thr Ala Asn Asp Val Met Val Thr Gly 20 25 30 Ile Ser Ile Ser Glu Lys Gln Val Glu Arg Ala Asn Asp Arg Ala Tyr 35 40 45 Lys Ala Asp Val Asp Asp Arg Val Val Phe Glu Tyr Ala Asp Ala Met 50 55 60 Glu Leu Pro Tyr Pro Asp Ala Ser Phe Asp Val Val Trp Ala Leu Glu 65 70 75 80 Ser Leu His His Met Pro Asp Arg Trp His Val Ile Arg Gln Ala Ala 85 90 95 Arg Val Leu Arg Pro Gly Gly Arg Leu Ala Leu Gly Asp Phe Leu Leu 100 105 110 Val Pro Ser 115 7 116 PRT Saccharopolyspora erythraea 7 Gly Ile Ser Glu Gly Asp Glu Val Leu Asp Val Gly Phe Gly Leu Gly 1 5 10 15 Ala Gln Asp Phe Phe Trp Leu Glu Thr Arg Lys Pro Ala Arg Ile Val 20 25 30 Gly Val Asp Leu Thr Pro Ser His Val Arg Ile Ala Ser Glu Arg Ala 35 40 45 Glu Arg Glu Asn Val Gln Asp Arg Leu Gln Phe Lys Glu Gly Ser Ala 50 55 60 Thr Asp Leu Pro Phe Gly Ala Glu Thr Phe Asp Arg Val Thr Ser Leu 65 70 75 80 Glu Ser Ala Leu His Tyr Glu Pro Arg Thr Asp Phe Phe Lys Gly Ala 85 90 95 Phe Glu Val Leu Lys Pro Gly Gly Val Leu Ala Ile Gly Asp Ile Ile 100 105 110 Pro Leu Asp Leu 115 8 120 PRT Artificial Sequence A consensus sequence 8 Xaa Xaa Xaa Xaa Gly Xaa Arg Xaa Leu Asp Xaa Gly Xaa Gly Xaa Gly 1 5 10 15 Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Thr 20 25 30 Gly Xaa Xaa Xaa Xaa Pro Xaa Xaa Val Xaa Xaa Ala Xaa Xaa Xaa Ala 35 40 45 Glu Xaa Ala Gly Val Xaa Asp Arg Xaa Xaa Phe Xaa Xaa Gly Asp Xaa 50 55 60 Xaa Xaa Leu Pro Xaa Pro Asp Gly Xaa Phe Asp Xaa Val Tyr Xaa Leu 65 70 75 80 Glu Ser Xaa Leu His Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Val Xaa 85 90 95 Arg Xaa Xaa Xaa Xaa Val Leu Xaa Pro Gly Xaa Gly Arg Leu Xaa Xaa 100 105 110 Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa 115 120 9 3765 DNA Streptomyces lavendulae 9 ggatccgagg gccggagtgg gattcggctc aatgaaccat gcacacagca cataccagga 60 cggtgtcgcg cccaccatac gcgacgcttc ccgctccctc cagccgtgcg gtttgagcca 120 cttcgacgcc ggataacgtt gccgacaggc ccgccgagca gcccctgaac tggatcaatt 180 cccttgggaa taaggcagtt tcactgctca accaccctgc tgacgagaat ccaccgccga 240 ccggcggtcg gggcagacct tcccggcaag ggtgttgact ccggcaactg ccctatggag 300 gctcgtgtct ggcatccgat cccggcctat gaccgggggc cggatcacat gcccgctccg 360 gccacccctc acaccgcggg ccggatttcc cgccgccccc gaggaacggc gtttcccgtc 420 gggtcacgca ccacccttcc cgacgcgggg cgaacacaac ggaaccgggc cgtgaagcca 480 cggccaccga aggcaaaggc ctcgacaccc gccctcccgc cgtacagcgc cccgaagtcg 540 accgtgccgc cgcacccgca ggaccgaaag gctgctcaat gacacctacg tccggtgatg 600 acgtcctgtc ctttccctca tggccgcaac acggcgcgga ggagcgcgcc ggactcctgc 660 gggccctgga ccagaagggg tggtggcgcg acgcggggca ggaggtcgat ctcttcgagc 720 gggagttcgc cgaccaccac ggcgccccgc acgcgatcgc cacgacgaac ggcacccacg 780 ccctggaact cgccctgggg gtcatgggga tcggccccgg tgacgaggtc atcgtccccg 840 cgttcacctt catctcgtcg tcgctggccg tgcagcgcat gggcgcggtg ccggtgccgg 900 cggacgtacg gcccgacacc tactgcctcg atgccgacgc ggcggcggcg ctggtgacgc 960 cacgcaccaa agcgatcatg ccggtccaca tggcgggcca gttcgccgac atggacgccc 1020 tggagaagct ctccgtcgcg acgggcgtgc cggtcctcca ggacgccgcg cacgcccacg 1080 gcgcgcagtg gcagggccgc cgggtcgggg agctcggctc gatcgccgcc ttcagcttcc 1140 agaacggcaa gctgatgacc gccggcgagg gcggcgccct gctcctgccg gacgacgagt 1200 ccttccacga ggcgttcctc cagcactgct gcggccgccc gcccggggac cgcgtctacc 1260 gccatctgac gcagggctcc aactaccgca tgaacgagtt ctccgcgagc gtcctgcgtg 1320 ctcaactgaa gcgcttgaag gatcagttgc gcatcaggga ggagcgctgg gcccagctgc 1380 gtacggcact ggccgccatc gacggcgtgg tgccgcaggg gcgcgacgag cgcggcgacc 1440 tccactccca ctacatggcc atggtccggc tgcccggcat ctcggcccgg cgccgcctcg 1500 cgctggtgga cgcgctggtc gagcggggag tgcccgcgtt cgtcggcttc ccgccggtct 1560 accgcaccga gggtttcgcg cgcggcccgg cgccggcgga cgccgaggag ctggccaaga 1620 gctgtcccgt ggcggaggag atcggcagcg actgcctctg gctgcaccat cgcgtcctgc 1680 tcgccgacgt gaccacgctg gaccggctgg cggaggtctt ctccggcctc gtcggcgcgc 1740 tctgacccga tgcgggcccc caacggcacc accgcccccc ggctgagcgt cgtcgtcccc 1800 agccgggggc ccgcggcacg cctgcgcgcg accctcgcat gccttgccgg cccctccccg 1860 ggaacgccgc ccttcgaagt ggtcgtcgtc gacgacaacg acgggggcga cgccggtgat 1920 caactgatcg ccgtgacagg cgagatgagc ggccttctcc cgctgcgcgt ggtgcgggga 1980 ccgctgcggg ggcgggccgc cgcccggaac gccggggcgg ccgcggccct cgcgccccgg 2040 ctggtcttcc tcgacgacga cgtcctggtg gggcccggct tcctcgccgc acacgccgcg 2100 gccgcggaac cggacgcctt cacccacggc cggctgcgcg aactccccac cgcggcgcgg 2160 ttcctcgccg ctgtcgagaa ggccgccccg accgaggtcc gccgcgcccg cgccggactc 2220 gaacccgctg ccccggccgc ctccgagcgg cgccaaccgc accggcggct cgtcgccaac 2280 gccctggagc gggccgtgga ggccatggcc ggcggctccc tgccggacgt cgccccctgg 2340 ctcggcttca tcggcgcgaa caccgccctc gacaaggccg catgggagca taccggcgga 2400 ttcgacgagg agttcgggct cacctggggg tgcgaggacc tggagttcgg cttccgcctg 2460 cacgccgccg ggctgcgcag gaccctcgcc cccgacgccc tcggtgtgca cctcagccac 2520 gcccgccccg gccgctggga gcagcaccac cgcaacctca cgcacttctc cgccggccac 2580 ccgcacccgt cggtacgcgc cttggaggcc ctgctcgggc ccgacggcac gccggaggcg 2640 tatgtgcgcg ccgtcctggc cgaagaggcc gcaccggcac gggacgcggc gcgatgagcg 2700 gcacaccggc caccgcgccg tacggtcccg tggtgctctc cccgcacgcg gacgacgccg 2760 tgtggtccct gggcgggcgg ctggcgcgct gggccgccga gggcccgcgg ccgaccgtcg 2820 tcacggtctt cgccgggccc gcggccggga agcccgagtc gtggcggagc gccgccgatc 2880 ccgcggtgcg ccgggccgag gaccgggcgg catgtgccga actgggcgtg cgccacgtgc 2940 cgctgggctt caccgacgcg gcactgcgta cggcctcggg cgcctatctc tacgcttccc 3000 cgcgccggct cttcggcccc tggcacccgg ccgacctccc gctgctggag gaggtgcggg 3060 cggctctgct gccgctgtgc gcgggggcgt cgagcgtcca cgttcccctg gcggcgggcc 3120 ggcacgtcga ccaccgcctg gtccgcggcg cggtggagcc cctgtccccc gcccgtaccg 3180 tcttctacga ggacttcccc taccggctgc gcgaacgtga ccacacgaac ctgcggccgc 3240 gcacggaacg gctgccgtcc gaggcggtgg accgctggct gaccgccgcc ggtcactact 3300 ccagccaggc gagcgcccac ttcggcggtg cggccgccct gcgcgaggcc ctgttcgccc 3360 gcgcccgcgc acacggcggg cccggccggc ccggccacgc cgaccgccac tgggtgcccg 3420 tcggccagga cgaccggggc gaggcccggc cggcacccgt ggaaaggggg ccgtgaccca 3480 cgccgtgcgc agccccacca cgagagaggc cactcatgtc ccgtagcacc cacccgccga 3540 cagccacccc cgacgcgggc accaggcgac gcctgccgct gatcggcaac gacctggtca 3600 tcaacgagga ctcctgcaac ctcagctgca cctactgcct caccggacag agcaacctca 3660 aggagggcca ctcccttcaa ctgatcttcg agcccccgcg gcgcgacagc tacgccaagg 3720 acagcgggct ggggcagcgc atggacaagg tcgccgaccg gatcc 3765 10 388 PRT Streptomyces lavendulae 10 Met Thr Pro Thr Ser Gly Asp Asp Val Leu Ser Phe Pro Ser Trp Pro 1 5 10 15 Gln His Gly Ala Glu Glu Arg Ala Gly Leu Leu Arg Ala Leu Asp Gln 20 25 30 Lys Gly Trp Trp Arg Asp Ala Gly Gln Glu Val Asp Leu Phe Glu Arg 35 40 45 Glu Phe Ala Asp His His Gly Ala Pro His Ala Ile Ala Thr Thr Asn 50 55 60 Gly Thr His Ala Leu Glu Leu Ala Leu Gly Val Met Gly Ile Gly Pro 65 70 75 80 Gly Asp Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Leu 85 90 95 Ala Val Gln Arg Met Gly Ala Val Pro Val Pro Ala Asp Val Arg Pro 100 105 110 Asp Thr Tyr Cys Leu Asp Ala Asp Ala Ala Ala Ala Leu Val Thr Pro 115 120 125 Arg Thr Lys Ala Ile Met Pro Val His Met Ala Gly Gln Phe Ala Asp 130 135 140 Met Asp Ala Leu Glu Lys Leu Ser Val Ala Thr Gly Val Pro Val Leu 145 150 155 160 Gln Asp Ala Ala His Ala His Gly Ala Gln Trp Gln Gly Arg Arg Val 165 170 175 Gly Glu Leu Gly Ser Ile Ala Ala Phe Ser Phe Gln Asn Gly Lys Leu 180 185 190 Met Thr Ala Gly Glu Gly Gly Ala Leu Leu Leu Pro Asp Asp Glu Ser 195 200 205 Phe His Glu Ala Phe Leu Gln His Cys Cys Gly Arg Pro Pro Gly Asp 210 215 220 Arg Val Tyr Arg His Leu Thr Gln Gly Ser Asn Tyr Arg Met Asn Glu 225 230 235 240 Phe Ser Ala Ser Val Leu Arg Ala Gln Leu Lys Arg Leu Lys Asp Gln 245 250 255 Leu Arg Ile Arg Glu Glu Arg Trp Ala Gln Leu Arg Thr Ala Leu Ala 260 265 270 Ala Ile Asp Gly Val Val Pro Gln Gly Arg Asp Glu Arg Gly Asp Leu 275 280 285 His Ser His Tyr Met Ala Met Val Arg Leu Pro Gly Ile Ser Ala Arg 290 295 300 Arg Arg Leu Ala Leu Val Asp Ala Leu Val Glu Arg Gly Val Pro Ala 305 310 315 320 Phe Val Gly Phe Pro Pro Val Tyr Arg Thr Glu Gly Phe Ala Arg Gly 325 330 335 Pro Ala Pro Ala Asp Ala Glu Glu Leu Ala Lys Ser Cys Pro Val Ala 340 345 350 Glu Glu Ile Gly Ser Asp Cys Leu Trp Leu His His Arg Val Leu Leu 355 360 365 Ala Asp Val Thr Thr Leu Asp Arg Leu Ala Glu Val Phe Ser Gly Leu 370 375 380 Val Gly Ala Leu 385 11 272 PRT Streptomyces lavendulae 11 Met Val Val Val Asp Asp Asn Asp Gly Gly Asp Ala Gly Asp Gln Leu 1 5 10 15 Ile Ala Val Thr Gly Glu Met Ser Gly Leu Leu Pro Leu Arg Val Val 20 25 30 Arg Gly Pro Leu Arg Gly Arg Ala Ala Ala Arg Asn Ala Gly Ala Ala 35 40 45 Ala Ala Leu Ala Pro Arg Leu Val Phe Leu Asp Asp Asp Val Leu Val 50 55 60 Gly Pro Gly Phe Leu Ala Ala His Ala Ala Ala Ala Glu Pro Asp Ala 65 70 75 80 Phe Thr His Gly Arg Leu Arg Glu Leu Pro Thr Ala Ala Arg Phe Leu 85 90 95 Ala Ala Val Glu Lys Ala Ala Pro Thr Glu Val Arg Arg Ala Arg Ala 100 105 110 Gly Leu Glu Pro Ala Ala Pro Ala Ala Ser Glu Arg Arg Gln Pro His 115 120 125 Arg Arg Leu Val Ala Asn Ala Leu Glu Arg Ala Val Glu Ala Met Ala 130 135 140 Gly Gly Ser Leu Pro Asp Val Ala Pro Trp Leu Gly Phe Ile Gly Ala 145 150 155 160 Asn Thr Ala Leu Asp Lys Ala Ala Trp Glu His Thr Gly Gly Phe Asp 165 170 175 Glu Glu Phe Gly Leu Thr Trp Gly Cys Glu Asp Leu Glu Phe Gly Phe 180 185 190 Arg Leu His Ala Ala Gly Leu Arg Arg Thr Leu Ala Pro Asp Ala Leu 195 200 205 Gly Val His Leu Ser His Ala Arg Pro Gly Arg Trp Glu Gln His His 210 215 220 Arg Asn Leu Thr His Phe Ser Ala Gly His Pro His Pro Ser Val Arg 225 230 235 240 Ala Leu Glu Ala Leu Leu Gly Pro Asp Gly Thr Pro Glu Ala Tyr Val 245 250 255 Arg Ala Val Leu Ala Glu Glu Ala Ala Pro Ala Arg Asp Ala Ala Arg 260 265 270 12 260 PRT Streptomyces lavendulae 12 Met Ser Gly Thr Pro Ala Thr Ala Pro Tyr Gly Pro Val Val Leu Ser 1 5 10 15 Pro His Ala Asp Asp Ala Val Trp Ser Leu Gly Gly Arg Leu Ala Arg 20 25 30 Trp Ala Ala Glu Gly Pro Arg Pro Thr Val Val Thr Val Phe Ala Gly 35 40 45 Pro Ala Ala Gly Lys Pro Glu Ser Trp Arg Ser Ala Ala Asp Pro Ala 50 55 60 Val Arg Arg Ala Glu Asp Arg Ala Ala Cys Ala Glu Leu Gly Val Arg 65 70 75 80 His Val Pro Leu Gly Phe Thr Asp Ala Ala Leu Arg Thr Ala Ser Gly 85 90 95 Ala Tyr Leu Tyr Ala Ser Pro Arg Arg Leu Phe Gly Pro Trp His Pro 100 105 110 Ala Asp Leu Pro Leu Leu Glu Glu Val Arg Ala Ala Leu Leu Pro Leu 115 120 125 Cys Ala Gly Ala Ser Ser Val His Val Pro Leu Ala Ala Gly Arg His 130 135 140 Val Asp His Arg Leu Val Arg Gly Ala Val Glu Pro Leu Ser Pro Ala 145 150 155 160 Arg Thr Val Phe Tyr Glu Asp Phe Pro Tyr Arg Leu Arg Glu Arg Asp 165 170 175 His Thr Asn Leu Arg Pro Arg Thr Glu Arg Leu Pro Ser Glu Ala Val 180 185 190 Asp Arg Trp Leu Thr Ala Ala Gly His Tyr Ser Ser Gln Ala Ser Ala 195 200 205 His Phe Gly Gly Ala Ala Ala Leu Arg Glu Ala Leu Phe Ala Arg Ala 210 215 220 Arg Ala His Gly Gly Pro Gly Arg Pro Gly His Ala Asp Arg His Trp 225 230 235 240 Val Pro Val Gly Gln Asp Asp Arg Gly Glu Ala Arg Pro Ala Pro Val 245 250 255 Glu Arg Gly Pro 260 13 386 PRT Streptomyces collinus 13 Met Ser Ser Gly Val Gln Leu Gly Ser Ala Phe Arg Val Trp Pro Gln 1 5 10 15 Tyr Asp Asp Ala Glu Arg Thr Gly Leu Ile Arg Ala Leu Glu Gln Gly 20 25 30 Gln Trp Trp Arg Met Gly Gly Gly Glu Val Glu Arg Phe Glu Arg Glu 35 40 45 Phe Ala Glu Tyr His Gly Gly Glu His Ala Leu Ala Val Thr Asn Gly 50 55 60 Thr His Ala Leu Glu Leu Ala Leu Glu Val Met Gly Val Gly Pro Gly 65 70 75 80 Thr Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln Ala 85 90 95 Ala Gln Arg Leu Gly Ala Val Val Val Pro Val Asp Val Asp Pro Glu 100 105 110 Thr Tyr Cys Ile Asp Pro Ala Glu Ala Ala Lys Ala Ile Thr Pro Arg 115 120 125 Thr Arg Ala Ile Met Pro Val His Met Ala Gly Gln Leu Ala Asp Met 130 135 140 Asp Ala Leu Glu Lys Val Ala Ala Asp Ser Gly Val Pro Leu Ile Gln 145 150 155 160 Asp Ala Ala His Ala Gln Gly Ala Thr Trp Asn Gly Arg Arg Leu Gly 165 170 175 Glu Leu Gly Ser Val Ala Ala Phe Ser Phe Gln Asn Gly Lys Leu Met 180 185 190 Thr Ala Gly Glu Gly Gly Ala Val Leu Phe Pro Thr Ala Glu Met Ala 195 200 205 Glu His Ala Phe Leu Arg His Ser Cys Gly Arg Pro Arg Asn Asp Arg 210 215 220 Gly Tyr Phe His Arg Thr Ser Gly Ser Asn Phe Arg Leu Asn Glu Phe 225 230 235 240 Ser Ala Ser Val Leu Arg Ala Gln Leu Ala Arg Leu Asp Gly Gln Ile 245 250 255 Arg Thr Arg Glu Glu Arg Trp Pro Leu Leu Ser Ser Leu Leu Ala Glu 260 265 270 Ile Pro Gly Val Val Pro Gln Arg Leu Asp Arg Arg Pro Asp Arg Asn 275 280 285 Pro His Tyr Met Ala Met Phe Arg Val Pro Arg Ile Thr Glu Glu Arg 290 295 300 Arg Ala Arg Val Val Asp Thr Leu Val Glu Arg Gly Val Pro Ala Phe 305 310 315 320 Val Ala Phe Arg Ser Val Tyr Arg Thr Asp Ala Phe Trp Glu Met Gly 325 330 335 Ala Pro Asp Leu Ser Val Asp Glu Leu Ala Arg Leu Pro Pro Leu Arg 340 345 350 Gly Leu Thr Thr Asp Cys Leu Trp Leu His His Arg Thr Leu Leu Gly 355 360 365 Thr Glu Glu Gln Met His Glu Val Ala Ala Val Ile Ala Asp Val Leu 370 375 380 Gly Ser 385 14 388 PRT Actinosynnema pretiosum 14 Met Gly Ser Ser Pro Asp Ala Gly Ile Asp Phe Pro Ala Trp Pro Gln 1 5 10 15 His Asp Asp Ala Glu Arg Ala Ala Leu Leu Arg Ala Leu Asp Gln Gly 20 25 30 Gln Trp Trp Arg Val Gly Gly Ser Glu Val Asp Glu Phe Glu Arg Glu 35 40 45 Phe Ala Glu Tyr His Gly Ala Gly His Ala Leu Ala Val Thr Asn Gly 50 55 60 Thr His Ala Leu Glu Leu Ala Leu Gln Val Leu Asp Val Gly Pro Gly 65 70 75 80 Thr Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln Ala 85 90 95 Val Gln Arg Leu Gly Ala Val Ala Val Pro Val Asp Val Asp Pro Asp 100 105 110 Thr Tyr Cys Leu Asp Val Ala Ala Ala Glu Asp Ala Val Thr Ser Arg 115 120 125 Thr Ser Ala Ile Met Pro Val His Met Ala Gly Gln Phe Ala Asp Met 130 135 140 Asp Arg Leu Asp Lys Leu Ser Ala Ser Thr Gly Val Pro Val Val Gln 145 150 155 160 Asp Ala Ala His Ala His Gly Ala His Trp Arg Gly Lys Arg Val Gly 165 170 175 Glu Leu Gly Ser Ile Ala Thr Phe Ser Phe Gln Asn Gly Lys Leu Met 180 185 190 Thr Ala Gly Glu Gly Gly Ala Val Leu Phe Ala Asp Gln Ala Gln Trp 195 200 205 Glu Lys Ala Phe Val Leu His Ser Cys Gly Arg Pro Lys Gly Asp Arg 210 215 220 Gly Tyr Phe His Leu Thr Ser Gly Ser Asn Phe Arg Met Asn Glu Phe 225 230 235 240 Ser Ala Ala Val Leu Arg Ala Gln Leu Gly Arg Leu Asp Ser Gln Ile 245 250 255 Ala Thr Arg Gln Ala Arg Trp Pro Val Leu Ser Ala Leu Leu Ala Gly 260 265 270 Ile Asp Gly Val Val Pro Gln Thr Val Asp Pro Arg Ser Asp Arg Asn 275 280 285 Pro Ser Tyr Met Ala Met Phe Arg Met Pro Gly Val Thr Glu Glu Arg 290 295 300 Arg Asn Ala Val Val Asp Glu Leu Val Arg Arg Gly Ile Pro Ala Phe 305 310 315 320 Met Ala Phe Arg Ala Val Tyr Arg Thr Gln Ala Phe Trp Glu Thr Gly 325 330 335 Ala Pro Asp Leu Thr Pro Glu Glu Leu Ala Ala Arg Cys Pro Val Ser 340 345 350 Glu Glu Ile Thr Arg Asp Cys Val Trp Leu His His Arg Val Leu Leu 355 360 365 Gly Ala Glu Glu Gln Val Arg Arg Leu Ala Ala Val Val Ala Asp Val 370 375 380 Val Ala Gly Ala 385 15 388 PRT Amycolatopsis mediterranei 15 Met Asn Ala Arg Lys Ala Pro Glu Phe Pro Ala Trp Pro Gln Tyr Asp 1 5 10 15 Asp Ala Glu Arg Asn Gly Leu Val Arg Ala Leu Glu Gln Gly Gln Trp 20 25 30 Trp Arg Met Gly Gly Asp Glu Val Asn Ser Phe Glu Arg Glu Phe Ala 35 40 45 Ala His His Gly Ala Ala His Ala Leu Ala Val Thr Asn Gly Thr His 50 55 60 Ala Leu Glu Leu Ala Leu Gln Val Met Gly Val Gly Pro Gly Thr Glu 65 70 75 80 Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln Ala Ala Gln 85 90 95 Arg Leu Gly Ala Val Thr Val Pro Val Asp Val Asp Ala Ala Thr Tyr 100 105 110 Asn Leu Asp Pro Glu Ala Val Ala Ala Ala Val Thr Pro Arg Thr Lys 115 120 125 Val Ile Met Pro Val His Met Ala Gly Leu Met Ala Asp Met Asp Ala 130 135 140 Leu Ala Lys Ile Ser Ala Asp Thr Gly Val Pro Leu Leu Gln Asp Ala 145 150 155 160 Ala His Ala His Gly Ala Arg Trp Gln Gly Lys Arg Val Gly Glu Leu 165 170 175 Asp Ser Ile Ala Thr Phe Ser Phe Gln Asn Gly Lys Leu Met Thr Ala 180 185 190 Gly Glu Gly Gly Ala Val Val Phe Pro Asp Gly Glu Thr Glu Lys Tyr 195 200 205 Glu Thr Ala Phe Leu Arg His Ser Cys Gly Arg Pro Arg Asp Asp Arg 210 215 220 Arg Tyr Phe His Lys Ile Ala Gly Ser Asn Met Arg Leu Asn Glu Phe 225 230 235 240 Ser Ala Ser Val Leu Arg Ala Gln Leu Ala Arg Leu Asp Glu Gln Ile 245 250 255 Ala Val Arg Asp Glu Pro Trp Thr Leu Leu Ser Arg Leu Leu Gly Ala 260 265 270 Ile Asp Gly Val Val Pro Gln Gly Gly Asp Val Arg Ala Asp Arg Asn 275 280 285 Ser His Tyr Met Ala Met Phe Arg Ile Pro Gly Leu Thr Glu Glu Arg 290 295 300 Arg Asn Ala Leu Val Asp Arg Leu Val Glu Ala Gly Leu Pro Ala Phe 305 310 315 320 Ala Ala Phe Arg Ala Ile Tyr Arg Thr Asp Ala Phe Trp Glu Leu Gly 325 330 335 Ala Pro Asp Glu Ser Val Asp Ala Ile Ala Arg Arg Cys Pro Asn Thr 340 345 350 Asp Ala Ile Ser Ser Asp Cys Val Trp Leu His His Arg Val Leu Leu 355 360 365 Ala Gly Glu Pro Glu Leu His Ala Thr Ala Glu Ile Ile Ala Asp Ala 370 375 380 Val Gly Arg Ala 385 16 1800 DNA Streptomyces lavendulae 16 atggaggacc gcaagcgcga ggggtatttc tagcgcggcg gggccggtgc ggcccacaag 60 cggaggacta gtccctaagt atgaagtccc ctactccgtt tgtctgttga gggcaggggc 120 gccgtctgag gatgatgcag tccatgtcac agttactttc cgggaaggac ggcgcccagg 180 aggcgccaag tcgcggcggg tccacgtggg tggcggtcct cgccgcgtgc gtggggcagt 240 tcgtggtggt cctcgacgtg tccgtcatca atgtcgcgct gccgtcgatc cgttccggcc 300 tcgacatcgg cgagacgggc ctgcagtggg tggtcaacgc ctacgtcatc gccttcgcgg 360 gcttcctgct gctcggcggc cgggcctccg acctcttcgg ccgcaaggcc gtgttcgtct 420 tcggcctcgg ggtgttcacc gccgcgagcc tgctcggcgg cctcgcgcag gcgccgtgga 480 tgctcatcgt cgcccgcgcc ctgcaaggca tcggggcggc cgtgctctca cccgccaccc 540 tcgcgatcct caccaccacg ttccccgagg gtccggcgcg catcaaagcc gtcgcgatct 600 ggacggccgt gggcacgggc ggcggcgcgg ccggcggcct catcggcggc ctgctcaccg 660 actacctctc gtggcgctgg gtgttgctga tcaacgtgcc gctgggcctt gtcgtgatcg 720 tcgcgaccgt cgcctggctg gccgagagcc gcagcgacca ggcacaccga cgccggctgg 780 acctcccggg agcggtgctg gtgaccctgg gcgtcggcag cctggcctac ggcatctcgc 840 agagcgaggg ccacggctgg ggctcgccgc ggacgctcac cttcctgatc gtcggtgtcg 900 tggcgctcct cgccttcgtc gccgtggagc agcgcacgcg cgagccgttg atgccgctcg 960 gtgtcttccg ggtgcgctcg gtgtcggcgg ccaacgccat caccatcgtc agtggcatgg 1020 gcttctacgc gatgtggtac ttcctctcgc tctacatgca gaacgtgctg aaatactccg 1080 ccgtacagac cggcctggcc ctgcttcccc acaccgccac catcatcctc tccgcgcagt 1140 tcgcaccccg cctgatgcgg tggatcaagg ggcgcaccct cctcgtgatc gcgggactgc 1200 tgaccgccgc gggcttcatc tggcagggga acatggacgc cgacggctcc ttcctggcga 1260 ccctgctcgg cccgggaatc gtcttctcct tcggcgcggg cctgatgatg acgctcctcg 1320 cggtctccgc cacgacgggc gtggagctct ccgaatcggg cctggtggcc ggcctcgcca 1380 acacctcgcg caccatgggc ggcgcgctcg gcctgtcggt cctcgcgtcc gtcgccgccc 1440 gccgcacggc cgacgtgggg cccggcgcgg agggcctggc ctccggctac ggtcgggcgt 1500 tcgtcgtgtc cggggccatc atcctcgtga gcatgctgat gatccccttc ctgcccaagc 1560 cccagcccca gaccccggcg gaatgacctg tgagcacgga catacgagga ggcttcgtgg 1620 ggcaggacag ccggccgcgg tggctcaccg acgaggaaca acgcgtgtgg cgcggctatc 1680 tgcgggccac caggctggtg gaggaccacc tggaccgccg cctccagcgg gaagcggaca 1740 tgccgcacct ctattacggt cttctcgtcc agctctccga ggccccgcgc cgggggatcc 1800 17 484 PRT Streptomyces lavendulae 17 Met Met Gln Ser Met Ser Gln Leu Leu Ser Gly Lys Asp Gly Ala Gln 1 5 10 15 Glu Ala Pro Ser Arg Gly Gly Ser Thr Trp Val Ala Val Leu Ala Ala 20 25 30 Cys Val Gly Gln Phe Val Val Val Leu Asp Val Ser Val Ile Asn Val 35 40 45 Ala Leu Pro Ser Ile Arg Ser Gly Leu Asp Ile Gly Glu Thr Gly Leu 50 55 60 Gln Trp Val Val Asn Ala Tyr Val Ile Ala Phe Ala Gly Phe Leu Leu 65 70 75 80 Leu Gly Gly Arg Ala Ser Asp Leu Phe Gly Arg Lys Ala Val Phe Val 85 90 95 Phe Gly Leu Gly Val Phe Thr Ala Ala Ser Leu Leu Gly Gly Leu Ala 100 105 110 Gln Ala Pro Trp Met Leu Ile Val Ala Arg Ala Leu Gln Gly Ile Gly 115 120 125 Ala Ala Val Leu Ser Pro Ala Thr Leu Ala Ile Leu Thr Thr Thr Phe 130 135 140 Pro Glu Gly Pro Ala Arg Ile Lys Ala Val Ala Ile Trp Thr Ala Val 145 150 155 160 Gly Thr Gly Gly Gly Ala Ala Gly Gly Leu Ile Gly Gly Leu Leu Thr 165 170 175 Asp Tyr Leu Ser Trp Arg Trp Val Leu Leu Ile Asn Val Pro Leu Gly 180 185 190 Leu Val Val Ile Val Ala Thr Val Ala Trp Leu Ala Glu Ser Arg Ser 195 200 205 Asp Gln Ala His Arg Arg Arg Leu Asp Leu Pro Gly Ala Val Leu Val 210 215 220 Thr Leu Gly Val Gly Ser Leu Ala Tyr Gly Ile Ser Gln Ser Glu Gly 225 230 235 240 His Gly Trp Gly Ser Pro Arg Thr Leu Thr Phe Leu Ile Val Gly Val 245 250 255 Val Ala Leu Leu Ala Phe Val Ala Val Glu Gln Arg Thr Arg Glu Pro 260 265 270 Leu Met Pro Leu Gly Val Phe Arg Val Arg Ser Val Ser Ala Ala Asn 275 280 285 Ala Ile Thr Ile Val Ser Gly Met Gly Phe Tyr Ala Met Trp Tyr Phe 290 295 300 Leu Ser Leu Tyr Met Gln Asn Val Leu Lys Tyr Ser Ala Val Gln Thr 305 310 315 320 Gly Leu Ala Leu Leu Pro His Thr Ala Thr Ile Ile Leu Ser Ala Gln 325 330 335 Phe Ala Pro Arg Leu Met Arg Trp Ile Lys Gly Arg Thr Leu Leu Val 340 345 350 Ile Ala Gly Leu Leu Thr Ala Ala Gly Phe Ile Trp Gln Gly Asn Met 355 360 365 Asp Ala Asp Gly Ser Phe Leu Ala Thr Leu Leu Gly Pro Gly Ile Val 370 375 380 Phe Ser Phe Gly Ala Gly Leu Met Met Thr Leu Leu Ala Val Ser Ala 385 390 395 400 Thr Thr Gly Val Glu Leu Ser Glu Ser Gly Leu Val Ala Gly Leu Ala 405 410 415 Asn Thr Ser Arg Thr Met Gly Gly Ala Leu Gly Leu Ser Val Leu Ala 420 425 430 Ser Val Ala Ala Arg Arg Thr Ala Asp Val Gly Pro Gly Ala Glu Gly 435 440 445 Leu Ala Ser Gly Tyr Gly Arg Ala Phe Val Val Ser Gly Ala Ile Ile 450 455 460 Leu Val Ser Met Leu Met Ile Pro Phe Leu Pro Lys Pro Gln Pro Gln 465 470 475 480 Thr Pro Ala Glu 18 990 DNA Streptomyces lavendulae 18 atcccgatcg tctcggacat gaccggcgac cttctcggcg cgcgggaggc ccaggacccc 60 gcctactggg tgtcccacat ccgccgcgcg gtgcgcttcc acgaccagat ccgccgtctg 120 cagcgctacg gggccggggc cttcgtcgag gtcggcccgg acacggtgct cagctcggcc 180 ggccaggcgt gcctgacgga ccaggcgggc aggagcgcgc ccgtcctggt gtccctcgcg 240 cacgccgagc gcgcggaggt gcccgcgctc ctgaccgctc tggccaccct gcacacccgt 300 ggcgtggccg tggactggcg ggcgtggttc ggcgacgggc cgcgcgcggc cggcctgccc 360 acatacgcgt tccagaagca gcactactgg ccgtcgggcc ccaccggttg gcggtccggg 420 cccgcccccg tacccctgcc ccaggccgga acggaggacg ccgaaaggcc cggtcgcgcc 480 gcggagtggc gggcgctgcc gcccggtgag cggtacgacg cgctgctgcg gatggtgcgc 540 ggcgaagccg ccgccgtgat ggggcacgcc gggccggagg cggtggagcc ggagcgcggc 600 ttcctcgacc acggcttcga ctcggtgatg gccgtgaagc tgcgcgaccg tctcgtggcc 660 gggacggggc gggagctgcc gacgaccctg ctgttcgacc accccacgcc cgcggccgtc 720 gccgactacc tgctggcggg gacgggcgag gccgagacgg cgccgtccgt gtccctgtcg 780 gaccagctcg accgcctgga ggccgacctc gcgcggctgc cggccgacga ccggcagcgc 840 gcccgcgtcg ccgagcggct caagggcctg ctcgcggtcc acgcgccgga ccggggcgcc 900 gggagcgagg acgcgccgga ccaggacgcg ctggacacgg cgaccgacga cgagatgttc 960 gagctgatcg agaaggaact ccgccgtgga 990 19 3978 DNA Streptomyces lavendulae 19 gtggatgaga ccaacgagac caaactccgc gagtacctgc ggctggtcac ggccgatctg 60 cggcgaaccc gcaggcagtt ggaggaggcc gaggacgcgg cccgcgagcc cgtcgcgatc 120 gtgggcatgg cgtgccgctt ccccggggac gtggcatcgc cggacgacct gtggcagctg 180 gtcgccgagg gccgggacgc cgtcaccgag ttccccgccg accggggctg ggacgtcgac 240 gccgtctacg accccgagcc gggcaccccg ggcaggacgt acgcgcgcca cggcggcttc 300 ctcaaggacg ccgccggatt cgacgccgcc ttcttcggca tcacgccgcg cgaggcgctc 360 gccatggacc cgcagcagcg catgatcatg gaggtctcct gggaggcgtt cgagcaggcg 420 ggcctcgacg cgaccaccct gcggggcgag gacgtcggcg tcttcgtcgg ctccaacagc 480 aacgactacc tgatcaacgt gctcgacgcg cgggacgtcg ccgagggctt catcgggacc 540 ggcaactccg ccagcatcct ctccggccgc gtcgcctaca ccttcggctt cgagggcccg 600 gccgtgtccg tcgacaccgc ctgctcctcc tcgctggtcg cgctgcacct ggccgcgcag 660 tccctgcggc agggggagtg ctccctggcg ctggcgggcg gcgcgacggt gatggccacg 720 ccgaccgcct tcatcgagtt cagccgccag cggggcctgg cccccgacgg ccgctgcaag 780 tccttctcgg cgaccgccga cggcaccacc tggtccgagg gcgcggccgt gctgctgctg 840 gcccggctct cggacgcccg ccgcctgggc taccccgtgc acgcggtcat ccggggcagc 900 gccgtcaacc aggacggcgc gagcgcgggc ctgaccgcgc ccaacggacc ggcgcaacag 960 cgggtgatcc ggcaggcact ggccaacgca cggctgacgg ccgacagcgt cgacgcggtc 1020 gaggcacacg gcaccggcac cccgctgggc gacccgatcg aggcccaggc cctcctcgcc 1080 acctacgggc gggcccgcgg cgagggcagg ccgctgtggc tgggctcgct gaagtcgaac 1140 ctgggccaca cccagtccgc ggccggcgcg ggcggcgtca tcaagatggt gatggccatg 1200 cggcacggga cgctgccccg cacgctgcac ctcacggagc ccaccccgcg cgtcgactgg 1260 tccgccggtg acgtacggct gctgaccgag gcccaggact ggccggacac cggacagccg 1320 cgccgtgcgg ccgtctcgtc cttcggcgtc agcggcacca acgcccatgt gatcctggag 1380 ggcccgcccg ccgaggaggc accggacgcg ccgctgccgg acgtctcctc gcagccgcgg 1440 ggcccgctgc cgtgggtcgt ctccggccgc agcgaggcgg ccgtccgagc gcaggccgag 1500 cgcctggcgg cccacctgac cgcgcgcccg cacctggcac cggccgacgt ggccaccgcg 1560 ctggccacca cgcgggcggc cttcgaccac cgggccgccg tcgtcggccg ggaccgtgag 1620 gaactgctcg ccggcctcgc ggccctggcc accggaaccc gcgcgcccgg cctggtcacc 1680 ggccggaccc cgccgtccgg cggcaaggcc gccttcctct tcaccggaca gggcagccag 1740 cagcccggca tgggccgcga actggcggct cacagcaccg tgttcgccga cgccctggac 1800 gaggtctgcg cccagctcga ccggcacctc gaccggccgc tgcgcgaggt gctgttcgcc 1860 gcggacggca cgcccgaggc cgccctgctc gacacgacgg cctacaccca gcccgcgctg 1920 ttcgccgtcg aggtcgcgct gctgcggctg ctggaggact ggggcttgcg gcccggcatg 1980 gtcgcgggcc actcggtcgg cgaactgacc gccgcctacg ccgccggggt ctggtcgctc 2040 gccgacgcct gcgccctggt cgccgcccgc ggccggctga cccaggcact gcccgcgggc 2100 ggcgccatgg tcgccgtgca ggcgaccgag gacgaggtgc gcgcccaact cgccgacggc 2160 cgccccggcg tggacatcgc cgccgtcaac ggaccggaag cggtggtgct gtccggcgac 2220 gaggccgccg tcacggacct ggcgcgcgag tgggccgccc gcggccggga gaccaggagg 2280 ctgcgggtca gccacgcctt ccactccgcc cacctggacg ccatgaccga ggcgttcgcc 2340 gaggtcgcac gaggggtgtc ctacagcgcg ccgtccctcc cggtggtctc cacgctcacc 2400 ggggcccccg tcaccgacga gctccgcagg ccggaacact gggtgcggca cgtccgggag 2460 acggtgcgct tccacgacgc ggtccgcgcc ctgcgcgacc gcggggccac cgcgttcctg 2520 gaggtcgggc ccggcggcgt gctgacggcc gcggcacgcc gatgcctgcc cgacgccgcc 2580 cccgagacgt tcgtccccgt gctgcggcgc cgcaggcccg aacccgagtc cgtgctgacg 2640 gccgtcgcgc aggcccacac gatcggcctc tcgccggcgt gggaccgcct gctgcccaag 2700 gcccggacgc gcgtggacct gcccacgtac gccttccagc gcggccacta ctggctggcg 2760 ggcatggccg gagcgggcac cgcgcggccg gtgcggccgg aagtgcagga gcccaccgcc 2820 ccctccggta cgccgccgct gtcgcgacgg ctggccgacg cgtcggagga ggagcgcggc 2880 cacctgctgc tgacgctggt acgcgagcag tcggccaccg tgatgggcgg cgtcgacccc 2940 gcgcaggtcg aacccgaccg ccccttcctg gagctcggct tcgactccct gatgggcgtc 3000 gagctgcgca ccgcgctcgc cgccgactgc gcactgcccc tgccgcccgg cctgatcttc 3060 gaccacccca cgcccgccgc cctggccgcc ttcctcggcg agcagctcgc ggcggcggcc 3120 tccggcaccc ccacggcggc ggcaccctcg ccgtactccc tggaggcgct gtaccgcaac 3180 gccaacaccc tcgaccggcc cgaggacgcg ctcgccctca ccaaggccgc ctcccggctg 3240 cgcccggtct tcgccagcgt ggccgaggcg gggcaggacc cggtcacggt ggagctggca 3300 caggccaccg gccttccggg cctgatctgc tgcccggcac ccgtgccgct gtacggggca 3360 cagcagtaca gccggctcgc agccgccttc cgcggcacgc gcggagtctc ggccctgctc 3420 gcccccggct tctccccggg cgaactgctg cccgccgact tcgaggtgat gcaggacttc 3480 ctcgccgagg gggtccggcg gcagaccgac ggcgcgccct tcgtcctcct gggccactcc 3540 tccgggggct ggttcgccta cagcctggcg gcccacctgg cgcgcaccgg gccgcgcccg 3600 gaggccgtcg tgctgctgga cacctatcag ctgcacgacc cggcgctgca ccgcatgcag 3660 cgcgaactcg cccagggcgt cctggaccgc gaggaggact tcggggcgat gacggacgta 3720 cggctgagtg ccatgggcaa atacttcgac ttcttcaccg actgggtggc cgaggacgcc 3780 ggtgtcccga cgctgctgct gcgggcctcc gagcctctgg gcgaggtcgt cgagggccag 3840 gagtggcgct ccacctggcc gttcgacagc acggtcctcg acacggaagg cgaccacttc 3900 gccatggtca acgaccacgc gccgcggacg gcccaggccg tgaacggctg gctgtcgggc 3960 ctcaccggcg gaaggggc 3978 20 570 DNA Streptomyces lavendulae 20 gtggagacac gcaacgccga acggccgtgg atacgcagct tccaccccgc tccccaggcc 60 cctgtgcggc tgctgtgcct gccgcacgcc gggggctccg cgagcgccta cttcgcgctg 120 tcgagggaac tggcgccccg ggtggaggtg ctcgccgtgc agtaccccgg gcggcaggac 180 cggcgcgacg agccgctgct ggactcgatc gaggccctgc gcgacggggt cgccgaggcc 240 ctgacgccct ggctggaccg gccggtcgcc ctcttcggcc acagcatggg cgccgtggtg 300 gcctacgagc tggcgcggct gctgtgccag gacgcgggcg tgccgctcac ccacctcttc 360 gtctccggac gccggggatc cgaccgaagt ctccgtcctt gccgccgtgt tccggaattc 420 accgtgacac cgccgcgcgg ctcttcttcc gaagtcctcc agatccggca cgagtttgta 480 tccgaacggg gttctgcgtg cgaaatactc tcttcgaatt gggtgacata cccccgatcg 540 gcaccgtacc cgagcagatg tacgcctcgg 570 21 1245 DNA Streptomyces lavendulae 21 gtgcgaaata ctctcttcga attgggtgac atacccccga tcggcaccgt acccgagcag 60 atgtacgcct cggtgatccg acgggagcgc tacggacagc cccaccaggc gttccgcagc 120 gaggtcgtgg acgtgccgaa ggtggggccc ggtcaggcgc tggtcctcgt gatggccgcg 180 ggcatcaact acaacaacgt ctgggcctcc ctggggcagc cggtcgacgt gatctccgcg 240 cggcagaagc agggccacag cgaggacttc cacatcggcg ggtccgaggg ctccggcgtg 300 gtgtgggcgg tgggggaggg cgtcacccag gtcgcggtgg gcgacgaagt gatcctctcc 360 ggctgccagt ggacggagac ggccgccgac atccggctcg gcgccgaccc catgacctcc 420 ggctcgcagt cggtgtgggg atacgagggc aactacggct ccttcgccca gttcgccctc 480 gtcgacgact atcagtgcca ccccaagccg cccggcctga cctgggagga agccgcctgc 540 ttcctgctca ccggggccac cgcctaccgc cagctgtgcg gctggcagcc gcacgacgtg 600 cggccgggcg acccggtcct catctggggc ggggccggcg ggctcggctc catggccatc 660 cagatcaccc gggcgcgggg cggcatcccc gtcgccgtgg tctccgacga ggagcgggcc 720 cgctactgcc gggagctcgg cgcccagggc accatcaacc gcctggactt cgaccactgg 780 ggacggctgc ccgacatcgg cgaccacgag gcgatgggcc gctggaccga gggtgtacgg 840 gccttcggcc ggcgcttctg ggaggtgctg ggcgagcgca ggtccccgcg catcgtcctg 900 gagcacagcg gccaggccac catccccacc tcgatgtacc tgtgcgacaa cgcgggcatg 960 gtcgtcatct gcggcggcac caccggctac aacgccgaca tcgacctgcg cttcctgtgg 1020 atgcgtcaga agcgcttgca gggctcgcac ttcgccaacc tgcggcagtg ccgcgacgtc 1080 atccacatgg tcgcgaacgg ccagctcgac ccgtgcctgt cgtggaccgg cggcttcgac 1140 gacatcggca aggcacacca gctgatgcac gacaaccagc acccccaggg caaccaggcc 1200 gtcctggtca acgcgccgcg gaccggcctg accaccttcg cctga 1245 22 1224 DNA Streptomyces lavendulae 22 gtgtccgaca ccgagcagca cgcgcccacg ctgccgcggc agcgcacctg ccccttctcg 60 ccgccgcccg agctcgagga gctgcggcgc accgatccca tcagcaggat gcggttcgcc 120 gacgactccc cgggatggct gctgacccgc cacgccgacg tccgcgccgc gctggccgac 180 cccggcgtca gctcgcaccc cggcaaggca ccccagccct ggcgcaacct cgcccccgag 240 atgcgcgccg agcactacct gccgggcttc ctgatcttca tggacccgcc ggaccacacc 300 cgctaccgcc gcctgctcac caagtggttc accatgcggg ccatccgcaa gctcgaaccc 360 aggatcgagc agatcgtcac cgagaccctc gacgccatgg aggcccaggg cggcaccgtc 420 gacctggtgc agtccttcgc gctgccgatc ccgctgctgg tcatctgcga gctgatgggc 480 atccgctacg aggagcgcga ggagttcatg gacatggtcc tgcgactcca ggccctggac 540 gccacgcccg aggaactcgg ggccctcggc gccaggatga acgagttcat gatgaagctc 600 gccgccgcca agcgcgcgaa ccccggcgac gacctgctca gccacctcgc ccacgacccc 660 gacgccgacc cggcgctcac ggatctggag atcgccggca tcggcgtgct gatgctcatc 720 gcggggcacg agacctcggc caacatgctg ggcgtcggca cctacaccct gctggagaac 780 gccgaccagt gggccctgct ccgtgacgac atcagcctga tcgaccgggc cgtcgaggag 840 ctgctgcgcc accagaccat cgtccagcag ggcctgccgc gcggcgtcac ccgggacatg 900 gagatcgccg ggcaccaggt gaagaccggg gagtccctgc tggcctcgct gcccgccgcc 960 aaccgcgacc ccgccgtctt ccccgacccc gaccgcctcg acatcacgcg cgagcacaac 1020 ccgcacctcg ccttcggcca cggcatccac ctctgcctgg gcatggagct cgcccgggtg 1080 gagatgcgcc aggcgtggcg cggcctcgtc acgcgcttcc ccggcctgcg catggccgcc 1140 gcgcccgagg acatccgctg gcgcgacgac cagatcgtct acggcgtgta caacctcccg 1200 gtgacctggg acgaggccaa gtga 1224 23 531 DNA Streptomyces lavendulae 23 atggacaagc tcgacatcct ctggagcgag cgcgagatcc gtgccgtgct gcagcgctac 60 tgccgcgggc tcgaccgcct cgacgaggaa ctggtcaagt ccgcctacca cgaggacgcg 120 cacgacgacc gcggcgtcat ccgcggcaac gcacacgact tcgtcaagca gatcgtcccg 180 ctcctgcgcg acgcctacac cggcaccctg cacaccctgc acggcagcac gatcgagatc 240 gacggggatg ccgcgggcgt ggagtcctac tgcaccgcct accactaccg cgagagcgac 300 ggcatcaagc gggtggagca gttcgccggg cgctacgtcg accgcttcga gcggcgcgac 360 ggcgtctgga agatcgcccg ccggctcgtg ctgaacgact tcagcctcgc ccaggaggtg 420 ccgctcgacc ccgccgaggc ccaggccggc ttcaacccct cccaccgcga cctcaccgac 480 gccagctacc aggtgctgcc gctgcgcggc ccggacgccc ccaccctctg a 531 24 1233 DNA Streptomyces lavendulae 24 gtgaccggcc ccgaggccgc ggtgcgcggg tgccccttcg gcgccggcga ggcgcccgcg 60 taccccttcc acgcccccga ccggctggag cccgacccgt actgggagcc gctgcgccgc 120 gagcggccgc tgcaacgcgt cacgctgccg tacggcggcg aggcgtggct cgccacccgc 180 tatcaggacg tgcgcgcggt cttcgccgac cgcaggttct cccggcagct cgccgtcgcg 240 cccggcgctc cgcgcttcct cccgcaccag ccgccgccgg acgccgtcct gagcgtcgag 300 ggccccgacc acgcgcggct gcgccggctg gtcgggaagg tcttcacgcc gcgccgcgtg 360 gaggacatgc gtccgctcat ccagcgcacc gccgacggac tcctcgacgc gatggaggag 420 atggggccgc ccgcggacct ggtcgaggac ttctccctgc ccttcgccgt gtccatgatc 480 tgcgagctgc tcggcgtgcc gcccgaggac cgcaagcggt tctgcgtctg gtcggacgcg 540 ctgctgacga ccaccgcgca cacccccgcc caggtgcgcg actacatgat gcagatgcac 600 gactacctcg gcgggctcgt cgcgcagcgc cgggtgcggc ccaccgcgga cctgatcggc 660 tccctcgtga ccgcgcgcga cgaggaggac aagctcaccg agggcgagct ggtgcggctg 720 gccgaggcca tcctcatcgc cggctacgag acctcggcga gccagatccc caacttcctc 780 tacgtcctct tccgccaccc gcagctgctg gagcggatca ggaacgacca cgacctcatc 840 cccgacgccg tcgaggaact gctgcgcttc gtgcccatcg gcaccgtgga cggctttccc 900 cgtacggcca ccgaggacgt cgagctcggg ggagtcctgg tcagggccgg ggagacggtc 960 gtgccgtcga tgggcgccgc caaccgcgac cccgagctgt tcacggaccc cgacgagctg 1020 gacctcgcgc ggcggccgaa tccgcacctg ggcttcggcg cgggaccgca ccactgcctg 1080 ggcgcccaac tggcccgggt ggagctccag atcacgctca cgacgctgtt ccgcagatac 1140 ccccgcctgc ggctggccgt gccggaggag agcctctcgt ggaaggaggg gctgatggtc 1200 cgcggcatgc acaccatgcc ggtcacctgg tga 1233 25 1107 DNA Streptomyces lavendulae 25 atgagcacca tcgacgaatg ggaacacagc acgaaggagg cgggcatgga ccccgcggcc 60 ctcagacgcc tgaccgatgt ggtgcgggcg aggggcggcg cggcgcagct gtgcgtcatg 120 cggcggggca ccgtggtcct ggaccgctcg ttcggctgct cctccgactc cctcttcctc 180 gtctacgcgg ccaccaagcc cgtcgccgcc ctcgccgtgc acgcgctcgc cgagcggggc 240 ctgatcgggc tggaccggcc ggtggccgaa tactggccgc agttcgcccg gcacggcaag 300 ggtgacgtga ccgtccgtca tgtcctccag caccgggccg gggtgccggt cggccggggc 360 atcgtgcgca cgatgcgcac cgccggcgac tgggagcgct ccgtgcgcga ccttgagcag 420 tcccggccca agtggcccgg cggcgaggtc gccgcctacc acttcatgag tttcggattc 480 attctcggcg aactggtgca gcgcgtcacc gggcggtcgt tccgagattt cgtgacttcc 540 gagctcttcg ccccacttgg gctgaatgat ttgcacatgg gattgcccgg cagtgcctgg 600 ccccggcatg tgcccgcgcg ggccgcccac ccctccgaat ggcccaatca gtggatgagc 660 aaccgccgcg gctaccgcca ggccgtcatt ccgtccgccg gtctttccgg aaccgccgca 720 caaatggccc gcttttacca gatgcttatg gagggcggct cgctcgacgg catccgcgtg 780 ctgcggcccg aaactgtgga ggaagccaga aaaccgtcca atgacggcgg aatcgacgct 840 tccctcaagc gtccggtccg ctggtcccac ggattcatgc tcggtggtcc gggcccggac 900 ccgcgggggc tgtccaatgt gctgggccgc acgagcgacc cgagcgcctt cgggcacgcg 960 ggcaccacgt ccagcgtcgt gtgggccgac cccacgcgcg agctggtcct cgcctacctc 1020 tccaacatcc agcccgagtt cggagcgggt atcgagcggc tccgcgaggt cagtgacctc 1080 gcgctcggtg cctgcgaggc aggctga 1107 26 858 DNA Streptomyces lavendulae 26 gtgctgaatc tgcccaaagg aatggagcgc gcgcatccgc attctccgcc acaggtggga 60 atactcggac ccttggaagt ccgctcggcc ggaggtgccg gaacgggagc cgcggtaagc 120 ggtattcgcg tacgcacatt gcttgccgcg ttgactgccc gcctggggca ggcgatgtcg 180 accgagcgca tcctcaaaga ggtctgggcc gacaacccgc ccgcgaccga tcgcaaggcg 240 gtggccgtcg ccgtcctgcg gctgcggcgg gtcctcggcg acaacgaagg acggtggctg 300 ctcacccgcc cctccggtta cgtcctggac atccccccgg accacctcga cgccgtacgc 360 gcggagaccc tggtgcggga aggccgggcc gccctggccg ccggcgaccc acgcgtcgcg 420 gcccgccacc tcacgcgcgc cctcgaccag tggcggggcg agccctacgc ggacgccaac 480 gccatctcga ccgtgtccca gcgcatcacg gagctggaga acctcaggtc cgaggccgtc 540 caggcgcaca tcgacgccag gctcgaactg ggtcaccacc aggaactggt cggcgaactc 600 cgctcgctga ccgccgcgaa ccccctgcac gagccgcact ggctgcagct gatgctcgcc 660 ctctaccgct ccggcaagca ggccgaggct ctcgccgcct atatgcagct gcggcaggcg 720 ctggccgaga acctgggcat cgacccgggt cgtcagctcc aggaactgca cctgcggatc 780 ctgcgcgccg acgcgggcct gctgacgggg tccgggccgg cggcaccggc cgagccactg 840 ctcgtacggc agtcctga 858 27 420 DNA Streptomyces lavendulae 27 atgcgtggat cgaaggccct ccgatacgcg gcccccgtcc tggtcgccgc cgcaaccggc 60 atcgccctcg ccgcgggacc ggcggccgcc gtcccgatcg gtcagtccgt gaacggcaag 120 atgacctact acaccgacca gggctacggc gcctgcggca cccccatcga cgcgaactcc 180 caggacctcg tcgcggtccc ggccgcgtgg tggacctccg ccaaccccaa caacgaccag 240 ctctgccagg gcatatcggt ggaggtcagc tacaacggca ggaccatcag agtgccggtg 300 cgggacaagt gcccttcgtg cgaccggacc cacatcgacc tcagcaggac ggccttccag 360 aagctggcgc cgctcgacag gggtgtggtc aacggcatca cctggaagtt cgtccgctga 420 28 2811 DNA Streptomyces lavendulae 28 atgagttcat caaatttaag gtcgcgggac tcttggaaca gatcaagacg acggagaaca 60 atgacgtact cccccggcgc gcggccgcgc ccggcccggc tgtccgcact gctgctcgca 120 ggcgcgctcg tcgcctcggt gccgcccgcg gccgccgcgc gagcgccgca accccccacc 180 gccgaccgcc cccgcaccgc cgcctccccc acaggcggct gccgtacggg tgacggctgg 240 acactcgact ccacccgcat cgaccccgac gacacccacc acgcctatgt cggcaacggc 300 tacctggggc agcgcgtacc gcccaacggc gccggctaca ccgacagcga caccaagacc 360 ggctggccgc tcttcgctcc ggcctacgac ggctcgttcg tgtccgggct ctacgcgcac 420 aacaagcaga ccgccgccga ccggcaggtg atcgccgctc tgcccacctg gaccggactg 480 gccgtcggca ccggcggcga gcacggcgat atcttcaact cttcgacgaa gtcgggccgg 540 atttccggat atcaccagac cctcttccag agctgcggca tcgtccgtac cgccctgacc 600 tggaccgccg ccgacggccg caggaccgac ctggtctacg aggtgctggc cgaccgcgac 660 gacccgcaca cgggcgccgt acggctgagc atgacgccgc gctggagcgg cgaggccacc 720 gtcaccgacc agctggacgg acgcggcgcg cggcgcatgc ggcagaccgg cggcggcgac 780 cgcaccggtg ggaccggccg ggacggccgc accatggacg tggccttccg caccgacggc 840 acggacaccg acggcgccgt cgcctccacc ctgagggccg ggcgcggtgt gcacacgacc 900 ggggaccgac gcgccgcggc cgcgaaggac ttgagcgtga accagtccct cacgttcccc 960 gtccgtgcgg gccacgcgta cgaactcacc aaatacgtgg gtgtcgacac cgcgctcacc 1020 tcgcacgcgc cccgcgagga cgccaccacc gcctccctgc gcgccgcccg ccgcggctgg 1080 gacgggctgc tgcgtgccca caccgccgcc tgggcccggc tgtggcgctc cgacatcgag 1140 ctgccgggac agcgcgacct ccaggcgtgg gtgcgttccg cccagtacgg gctgctgtcc 1200 agcacccggc agggggcatc caacagcatc gccccggccg ggctgaccag cgacaactac 1260 gcgggcctgg tgttctggga cgccgagacc tggatgtacc cggccctgct ggccaccgcg 1320 ccccaactcg ccaggaccgt cgtcgactac cgctaccgca ccctcgccgg agcgcgcgag 1380 aacgcccaca agctcggcta ccaagggctc ttctacccct ggaacagcgg cagcgagggc 1440 gacctggccc aggagtgcca cagcgtcgac ccgccccact gccgcaccca gatccacctc 1500 cagtcggaca tctccctcgc cacctggcag ttctacctcg ccaccggcga caccgcctgg 1560 ctgcgcgagc gcggctggcc ggtgatggag ggcatcgccg aattctgggc cgggcgggtc 1620 acccccaacg ccgacggcag ctactccatc aaggacaccg ccggccccga cgaatacagc 1680 aacggcgtcg acgacgcggt cttcaccaac gccggtgccg ccaccgccct gcgcgacgcc 1740 gcccgtgccg cgcggctgct gggcgagcgc gccccggcgg agtggacgac gatcgccgac 1800 cggatccgca tcccgtacga cgcgcggcac aaggtcttcg agcagtacga cggctacccg 1860 ggcagcaaga tcaagcaggc cgacacggtg ctgctgatgt accccctgga gtggccgatg 1920 tcccaggccg acgcggcgcg caccctcgac tactacgccc ggcgcaccga ccccgacggc 1980 cccgccatga cggactcggt ccacgccatc gacgccgcgg ccacgggcga gccgggctgc 2040 tcggcgtaca cctatctcca gcgttccgtc cggcccttcg tgcgcggtcc tttcgaccag 2100 ttctcggaag cccgcggcac caaggccggc gccgacgacc ccctggccgg ctcgcccgcc 2160 cacgacttcc tcaccggcaa gggcggcttc ctccagatct tcaccaacgg cctgaccggc 2220 atgcggatgc gcgaggaccg gctgcacctc gacccgatgc tgcccccgca gctcggccgc 2280 ggcgtcaccc tgcgcggcct gcactggcag ggccgcacgt acgacatcgc catcggcgcc 2340 cacgagacca ccgtgcggct caccgggggt gcgcccatga ccctctacac cccgcagggc 2400 gagcacgtgc tgaccaaggc ggcaccggcc gtgctcaaga cccgccgccc cgacctcgct 2460 cccaccgaca acgtggcccg ctgcaccacc gccggtgcct cctccgagga acccggtatg 2520 tacgcggcag ccgcggtcga cggcaacccc gccaccgcct gggtccccga cgggccgaac 2580 ggtgaactga ccaccgacct cggcaagtcc gtacgcgtca ccaaggccac ccccgtctgg 2640 agcggcccgg caccggcctc gtacagcgtc cagctctccc tcgacggccg gcactggcac 2700 gacgcggtcg cgggcggcgc tccggtgtcc gcgcggtacg tacgcgtcgc gctacgcggt 2760 caggccgatg ccaagtcccg tacgggcatc gccgagctga ccgttacgta g 2811 29 813 DNA Streptomyces lavendulae 29 atggaattcc tcgggccggc ggccggtgtc tcgggcgcca cgcggctgta cgcggtgctg 60 ggtgatcccg tcgcccaggt caaggcgccc ggtctgctca accccctgct gagcgaaagc 120 ggtctggacg ccgtggtggt gccggtgcac gtccgggcgc gggatctcgc cgaggtggtc 180 gaggggctca agcggatcgg caatctggac ggtctgctgg tcaccgtgcc gcacaaggcg 240 gccctgtgcg ggctcgcgga cgggctcggg ccggcggccg ccctcatcgg gacggccaac 300 gcgatgcggc gcgaacccga cggccgctgg tacgccgaga acttcgacgg gctcgggttc 360 gtccagggtc ttcaggcggc cgggcacacg gtgcgcgaca ggcatgtggc actggtcggc 420 gccggagggg cgggcagcgc gatcgccacg gcgctgctga tggccgacgc cgcgcgggtg 480 tccgtgcacg acaccgaccg cgcccagctc gacgcgctgc tgctgcggct cgggtcccgc 540 cggccggacg ggatccgggc gctggggccc ggcgatctgg aggcggccga tttcgccgtc 600 aacgcgacgc ctctgggcat gcgttccgag gacccgctgc ccttcgaccc cgcgagggtg 660 cgaccggatg ccgtggtggt cgacgtcgtc atgaagccgc acgagacggc gctgctgagc 720 gcggccgcca ccgccgggcg ccgtgtgcac cacggcatcc atatgctgga gcagcaggtt 780 ccgtgctacc gcgcgttctt cgggtggccg tga 813 30 948 DNA Streptomyces lavendulae 30 gtgacggggg acaccgacgg tgcgggcggc ggcgacgtga cgttccgctg gcccgccgcc 60 ggcgacgtca ccgcggatct ggacctgctc gccgcgcggg tccgcggtct tctgggacac 120 cgcgaggacc ccctcgccgg ggtcggcgtg gccatgcccg cgatctgcga cgcggccggg 180 acggtccgca cgtggccggg acggccgagc tgggcgggcc tgaacctgac ggccgccttc 240 gggcagttgc tgcccggcac cccggtcgcc tgcgccgacg acggtgacct ggccgcgctg 300 gcggagtccc gcgccgccgg ctgccggcat ctgctgtacg tgggggtcgg cacgggcatc 360 ggcggcggca tcgtccatga gggccgcgcc tggccgggcc ccggacgcgg ctcgtgcgag 420 gtcggccatg tcgtcgtcga ccgctcgggc ccacgctgcg actgcgggcg cgccggctgc 480 gtccaggcgg tcgcgtcggg accggcgacc ctccggcggg ccgccgaacg gcgcggccgg 540 gagaccggct tcgacgaact ggcctccggg gcgcgcttgc acgccccgtg ggcggaagcg 600 gccgtcgacg agagcgccgc ggccctggcc accgccgtga ccggcatctg cgagctggcc 660 caccccgaac tcgtcctcgt cggcggcggg ttcgcggcgg gcgtgccggg atacgtggcc 720 tcggtggcgg cgcacgtcga gcggctgacc cgcccgggaa cggatcccgt gcgggtgcgc 780 ccggcggtgc tcggcgggcg gtcctccctg cacggcgcac tgctgctcgc gcgggaggca 840 cacgggcggg gaaaccggcc gccggagagt gaccgtgttt cttccgatgt ttcttccgat 900 gtttctttcg ggggagtgac agacagggcc gttggccggt ccgactga 948 31 1545 DNA Streptomyces lavendulae 31 atgctcgaca ggcggagcgt cattcgcgtc ggcgccgggg tggcggcggc cgccgccgtg 60 gccggtacgg ccgccaccgg tgcggcggcc gtggggctgc cgggtgtacg gggacgcgcg 120 gcgtcgcgcg gggtcgactg ggcctcctta cgccgtcatc tgtcgggcga gctcgtcctg 180 ccggcggaca ccggatacga gcgggccagg aagctctaca gcggccagtt cgacggcatc 240 cgcccgcagg ccgtcgccta ctgccggacc gaggaggacg tgcggacgac cctcgcgttc 300 gcccaggacc acgcgctgcc cctcaccccg cgcagtggcg ggcacagctt cggcggctac 360 tccacgaccg acggaatcgt cctggacgtc tccggcttcc acgcggtgag cctcacccgg 420 aacaccgtcg tcatgggcgc gggcacccag caggtggacg ccctcaccgc cctgtcgccg 480 cgcggtgtcg ccgtggcgag cggcaactgc gcgggcgtct gtcccggcgg cttcgtccag 540 ggcggcggac tgggctggca gagccgcaag ttcggcatgg cgtgcgaccg gctcgtctcc 600 gcccgggtcg tgctcgccga cggccgcgcc gtgaccgcct ccgccaccga acaccccgac 660 cttttctggg cgatgcgcgg cggaggcggc ggcaacttcg gcgtcgtcac cggcttcgag 720 ctgcgcccca ccgacgtccc ctccgtcgtc agctacaacc tcacctggcc gtgggagtcg 780 gcgcggcgcg tcatcgaggc gtggcagcac tggatcatcg acggcccccg cgacctcggt 840 gccgcgatgg ccgtgcagtg gcccgacgcc gggaccggca cgccggtcgt ggtcgtcacc 900 ggcgcctggc tgggcgcggc cgacgcgctc acccccgtgc tggactccct ggtggcctcc 960 gtgggcagcg cgcccgccac ccgctcggcc aaggcgctct cccagcacga cgcgatgatg 1020 gcgcagtacg gctgcgccga cctcacgccc gagcagtgcc acacggtcgg ctactcgccc 1080 gaggccgcgc tgccccggca gaacttctcc atggaccgca accggctctt ctcccgggcc 1140 atcgggcaag gaggcgtcga gcggatcctg gaggcgttcg ccgccgaccc gcgcgccgga 1200 cagttccgct tcctgagctt cttcgccctc ggcggcgccg ccaaccgccc cgaccgcacc 1260 accaccgcct acgttcaccg cgacaccgag ttctacctcg gtttctcgat cgggctgaac 1320 gacccggagt acacggcgga ggacgagagg ctcggccgcg cctgggccgc gcgaggactg 1380 cgcacgctcg atccccactc caacggcgag agctaccaga acttcatcga cccggagctc 1440 gacgactgga agtcggccta ctacgccgag aactacgtgc gcctggccgc cgtcaaggcg 1500 gcctacgacc cgcaccggct cttctccttc gcgcaggccg tctga 1545 32 495 DNA Streptomyces lavendulae 32 gtggagaggg tggagctgat ccgctggccg gtggagtccg agcggcggga gcgctgccgc 60 gaccggggcg tcatgcggat cctggtgctg gaggcggggg ccgaggcacc cttgtgcgtg 120 gaccccaagg aggactgggt ccgcgctccc gtcagcaccg acgacctgcg ggcccgcgtc 180 gaggccctgc gccttcgggg agccgccgcc gagtcccggc ccgaggtcga cccgaacgga 240 gtgctgcgtt tccggtggcg ctccgccctg ctctcgccca ccgaggcccg gctcgtcgcc 300 cggctcgccg agtcctatgc cgaggtcgtc gcccgcgacg acctgctccg cccgcccccg 360 ggccgtaccg tgccgagccg taacgcgctc gacctccaca tcatgcggat ccgacggcgc 420 ctcgccgcgc tgggcctgag ggtgcgcacc gtccgggggc gtggctacgt cctggagagc 480 gcggaaggag tctga 495 33 1032 DNA Streptomyces lavendulae 33 gtgcagcagc ctcatcacag ccgcgtcgac gtggaactgg gcgagaggtc ctaccccgtc 60 cacgtcggac cgggggtccg ccacctcctg cccggcatcg tcgcctccct cggcgcgcac 120 cgcgccgccg tcgtgaccgc acggcccccc gacctggtgc ccgatcccgg cgtgcccgcg 180 ctgatcgtgc gggcacgtga cggcgagcgg cacaagacgc tcgccaccgt cgaggacctg 240 tgccgcaagt tcaccacctt cggcatcacg cgccacgacg tcgtcgtctc ctgcggagga 300 ggctcgacga ccgacaccgt cggcctggcg gcggcgctgc accaccgtgg ggtgccggtg 360 gtgcacctgc cgaccaccct cctggcccag gtggacgcga gcgtcggcgg caagacggcg 420 gtcaacctgc ccgagggcaa gaacctcgtc ggcgcctact ggcagcccaa ggccgtgctg 480 tgcgacacca cgtatctcca gacgctgccc gccgaggagt gggtcaacgg ctacggcgag 540 atagcgcgct gccacttcat cggtgccggc gacctccgcg gcctcgccgt ccacgaccag 600 gtcaccgcga gcctgcggct gaaggcgtcc gtcgtcgcgg ccgacgagcg ggacaccggc 660 ctgcggcaca tcctcaacta cggccatacg ctgggccacg cactggagac cgccaccggc 720 ttcgggctgc ggcacggact cggcgtggcg atcgggacgg tcttcgcggg ccggctcgcg 780 gaggcgctgg gccgcatcgg cgccgaccgc gcgcgggagc acaccgaggt cgtccgccac 840 tacggacttc ccgacagcct cccgggaaac accgacatca ccgagctcgt cgcgctgatg 900 aggcacgaca agaaggccac gtcgggactg accttcgtgc tcgacgggcc ttccggcgtg 960 gagctggtgt ccgggatccc ggaggacgtc gtcctgcgta cgctcgcggc gatgccgcga 1020 ggaacggcct ga 1032 34 441 DNA Streptomyces lavendulae 34 gtgttccgtc ttccgagggg aagtgaccgt ttcgtgtcgg cagagctgtc agaaccgctg 60 aagaaggccc tggactccct ggtgttcggc gtcgtggcga cgaccgaccc cgacggccgc 120 ccgcaccagt cggtggtgtg ggtccggcgc gagggctccg acgtgctgtt ctcgatcacg 180 cgcggcagcc gcaaggagag gaacatcctg cgcgacccgc gtgtgagcgt gctgatcagc 240 ccggcggact cgccgtacac ctacgccgcg atccggggca ccgcgcactt cgaggacgtg 300 ccggacccgg gcgcgtacct cgacacgttc tccataaagt accacggcgt gccctaccgg 360 gagtcgttcc ccgagccgcc ggaggtgagc accattctcg ccgtccggct cgttccgacg 420 tcggtctacg agcagtggtg a 441 35 828 DNA Streptomyces lavendulae 35 atgacggaaa ccgcgtccgc ctccgaccgg atggtcgaac tctacaaccg cgtcaccgac 60 ttgatggtgc acgcggaagg cggctacatg cacggtggct actgggcggg acccgacgtc 120 cccacgacgg tggaagaggc aggcgaccgg ctgaccgact acgtctcgga gcgcctgcgc 180 ctcgcccccg gggagcgggt gctcgacgtg gggtcgggca acggcaaggc caccttgcgc 240 atcgccgccc ggcacggggt gcgggccacc ggggtctcca tcaaccccta ccaggtgggt 300 ctgtcgcggc agctcgccga gaaggagggc gacgaggcga ccgagttccg catcggtgac 360 atgctcgcgc tcccctttcc cgacggctcg ttcgacgcct gttacgcgat cgagagcatc 420 tgccacgccc tggaacgggc cgacgtcttc accgagatcg cccgggtgct gcgcccgggc 480 ggccgggtga cggtgacgga cttcgtgctg cgccggcccc tgagcgacgc gtccaggacg 540 atcgtcgaca ccgccaacga caacttccag cagggccccg tcctcacccg cgaggcgtac 600 gaggactgca tgcggtcggt ggggctggag gtggtggagt tcctcgacat cggggacgag 660 gtgcggccct cctacgaggc ggtggcggcg aagatgcgtg cggccaggga cgagctcggc 720 tcccacatgg acgacgaggc gttccaccgc atggtcgacg gcatcgaccg catgggctcg 780 gtggaggagg tcggctactc ggtggtcacc gcgcggaaac cggcgtag 828 36 852 DNA Streptomyces lavendulae 36 atgccgcact ccgagctgtc cgaactcccc atgccctcac ccgcctccga ggaagtgggc 60 gcgctctacg accggttcac cgcgctggga gccgcctccc tcggcgagaa cctgcacttc 120 ggctactggg actcccccga cagccaggtg ccgctggccg aggccaccga ccggctcacc 180 gacatgatgg ccgagcggct gcgcatcggc gccggctccc gcgtcctgga cctcggctgc 240 ggcgtgggga ccccgggcgt acgcatcgcc cggctcagcg gagcgcatgt cacgggcatc 300 tcggtgagcc atgagcaggt cgtccgggcc aacgcgctgg ccgaggaggc cgggctcgcc 360 gaccgggcgc gcttccagcg ggccgacgcg atggacctcc ccttcgagga cgagagcttc 420 gacgccgtca tcgccctcga atcgatcatc cacatgcccg accgcgccca ggtgctcgcc 480 caggtcggcc gggtgctgcg gcccgggggc cgtctggtgc tcaccgactt cttcgagcgg 540 gcccccctcg cccccgaggg gcgggccgcc gtccagcgct acctccacga cttcatgatg 600 accatggtca gcgccgaggc gtaccctccc ctgctgcggg gggcgggcct gtggctggag 660 gagttcctcg acatcagcga ccagaccctg gagaagacct tcaggctgct ctcggagcgc 720 atcaactcct cgaagcagag gctggagacg cagttcggcg aggagatggt gaaccagttc 780 gaccccggcg acctcgtcgg cgtcaaggag ttcggctatc tgctgctggt cgcccagcgc 840 ccgggaaagt ga 852 37 1563 DNA Streptomyces lavendulae 37 gtgctgaaca ccctgtccac cgcgccgttc ctgtccacgg cctggctcgc cggggccgcg 60 aggctcgaac gcccgcccgt gggcgaacgc ggcacggtcg cgctccgcct ggagctcacc 120 gacccaccgc ccggcgaacc cccggccgtc gacgtccagg tggacctcgt cgccgggcgg 180 ctcggcctcg cggccgcggc cggtgagagt ccgggactgc ggatccggct tcccctggag 240 gccgcccgcg ccctgctgct cggccccgcg cgggatcgga ccggcgtatt cgagcggggc 300 gacgtacggg ccgagggcaa tttcagcctg ctgttcttca tcgacgccgc actggagcgg 360 gacgcctcgg gccatgtggc cgcgctcagg ggcacgcccg gtaccacggc gcgggaagcg 420 gccccgccgc ccggcaccga ggacgcggcc gaggccgtcc ggcgcgcccg tgcggcgctt 480 cccggcacca tgcgggagct ggagcgcgag gtcggcacct cgaccccggg ggcgcagatc 540 tacgtctccc gcgacggagt ccctctggcg gacgccgggt tggggctggc ccgccccggg 600 gtggcgatga cccaccggtc gctgcccctg tggtactgct gcgccaagcc actgctgtcg 660 gtcgccctgg gccggctgtg ggaggcggga gcgtacgacc cgtatctgcc cgtcgcgcac 720 tatctgccgg agttcggcaa ccggggcaag gagtccatca cctcgatgga actgctgacg 780 catacgggcc cgctgcccac cggcgacgac ccgctgcacg gcatcgtggc cggcccggac 840 gaggagcgtg tgcgccgtgc cttcgaggtg ccggtggcac cgcgtccggg gggcacgccc 900 ggcatcaact acagccagtg gtgggcctgg ttcgtcctgg cgcgcatcct tccggtcgtc 960 gacggcaggg agtaccgcgc gtacgtccag gaggagatcc tcgggccgtg cggcatgtcc 1020 ggcacccgtg tccacctgga tcgcgaggag ttcgccgcgc tcgggggcga gctgccgctg 1080 atccatgtga gcaaccccga gggcggcccg ctgcccaccc actggtggtc gacggaggcg 1140 gccaccacac gctgcatccc gggggtcaac acccgtggcc cgctgcggga catgggcagg 1200 ctcttcgaga tgctgctgcg cggcggggac gctcccggcg ggcgcgtcct ggcgccgccc 1260 accgtcgccg ccctcacggc ccggcaccgc accggcctcc aggaccgcta cggcaacgcc 1320 gactggggca tggggttccg cctcgaatgc cgtcagctgg atccgcggtt caccagcttc 1380 ggctcgtacg cctccccccg gtccttcggg cacgacgggc tgtggaccgc cgtggtcttc 1440 gccgacccgg acgccgctct cgtcgtcgcc ctccacctca acgggaaggt ggagcacgaa 1500 cggcaccgcg agcgcatcgt ccgcctcgcc gacgccgtct accaggacct ccgtctctcc 1560 tga 1563 38 1041 DNA Streptomyces lavendulae 38 atgacgcccg cgaccccgcg ctggagcgtc gtcgccccgc agggcaccaa cctcgaactg 60 gccggtacgg gcggccgcga gggctggcgg ctcctcctgg agaccgcccg caccgtccac 120 cggcacggcc gtggcgccct ctggctgctg gaccgcaccg acaccctgcc ccggcgcgag 180 cccgagccgg tctgggaggg ctggacggcg ctggcggccc tcgcgggcgc ggtgcccggt 240 ctggatctgg gactgctctc ctcggccccg ccgttccgca acgccgcgct gatcgccaag 300 cgggccgcga ccctggacgt cgtctgcgac ggccggctca ccctcggctt cccggcccgc 360 gagtacctgc cggagcacca ctcgacgggg cgcgaggtgc ccacgggcct ggaggcggac 420 gaggaggagg ccgccggcca ccgggctctc ggcgagacgg tcgaggccct gcgcgcgctg 480 tggggcggac agcccgtcac cttcaccggg gaacacatcc gcctcacttc ggcgcactgc 540 gtgcccgccc cacggcagca gcccctcccc ctcgcgctgc gcaccccggc cggggacgcc 600 gggagcggcg cgctgcggcc cgccgacgcc accgtgcggg agtgcgctca tgtccagtgg 660 accggtgagc ccgctcaggt cgccgcggcc gtcaccgcgt tccgccgccg tcgcacggag 720 ctcgggctcg atccggacgg cgtccggcac gcctgggccg cggagtgccg gatcttcgac 780 tccgtcctgg aacgcgaccg ctggctctcc accccgcacg aggtgctgtt ctggagccac 840 catcccgacc tgctggcgcg gcgcagcctg tacgggacgc cggaacagct caccgagcgc 900 gcccggcgcc tggtcgccgc gggcgtggcg gagttcgtgc tgtggttccg cgactacccg 960 gccaccacca gcctggagcg gctgttccag gaggtcgtcc cccaggtggc gccgggggcc 1020 gccaaggaag cggaggagtg a 1041 39 708 DNA Streptomyces lavendulae 39 atgcccctga accccccgcc cgcctcgcgg gccgcggcgg acgcgcccgc caccgctctc 60 ccgtgccggt tcaccaccgt ggtcttcgac ctcgacggcg tcctcatcga cagtttcgcg 120 gtgatgcgcg aggcgttcgc cgtggcctac cgcgaagtgg tggggccggg cgagccgccc 180 ttcgaggagt accgcacgca ccagggccgc tacttcccgg acatcatgcg gctgatgggc 240 ctgcccggcg agatggagga accgttcgtc cgggagagcc accggctgat ggaccgcgtc 300 gaggtgtacc cggacgtgcc gcagttgctg gcggagctgc gcgcggacgg cgtcggcacc 360 gcgatcgcca ccggcaagtc cggctcccgg gcgcgcgccg tgctggaggc ggtcgggctg 420 ctgcccctgc tggacgaggt ggtgggcagc gacgaggtgc cgaggcccaa gccgcacccc 480 gacatcgtgc gggaggcact gcgccggctg gacgcggcgc ccgaggacgc ggtcatggtc 540 ggcgacgcgg tgatcgacat ccgcagcggc cgcgccgccg ggaccgccac cgtgggcgcg 600 acctggggcg agggcgcggc cggccaactg cgcgccgagc ggcccgactt cctgctggac 660 aagccgcaga gcctgctcgc gctggtccgc agcggcggcc acgcatga 708 40 873 DNA Streptomyces lavendulae 40 gtggagcgtc tgaagctcgt gcccgacgag caccgccgtt tcaccgtcga cgagcagagc 60 gcgcgccggc tgcaccggat cggaccggag ctgctgtccg cgctgtgcga ggcgggcgtg 120 cccttcgtgg gaagcggcgc gggacgcctc ttcgacggct acgacctggg caacgcggcc 180 ctgcaccttg gcctgtcctc ggtgcagcgc cgggccatcc gctcgtgggc cggttccctg 240 cggaccgcct cggccgcgga gagcccgcgc tggcgcgtcg acgtcacggc gtcctgcccg 300 gtgcccggcc acgcgggccc gtgccgctac ggagtgctgc tgcccggcgc ccgccgcccg 360 gtggaggcgg cttcgccgcg ggagaccacg ctggcgcggc tgtacacacg gtcgcgcggc 420 cactggccgg acttcccccc ggccgtcctc gacctgctgc gcaccctgga gccggtcggc 480 ttcttcctgc tgcccgaagc gatccgctgg gacccggggt tcctgtggag cacgcacatg 540 gccgactgcg gcggcgccgc ggcctggctg gtggcggagg gccggcggcg cgggctcgac 600 gtgcggttct ccttcgggct gctggtggcc aagccgtact ccacaccgca ctgctgggcc 660 gagttcctgg tgggcggccg ctgggtgccg gccgatccgc tgctgctgag ggccatggcc 720 gcctggggcg ggctggacgc ggcggcccac ccgccgcaca gctcgccggg ggccgtctac 780 caccggctcg cgggccgctt cacgaaagtc gtcagccacg ccggggtctg ggccccgacg 840 tccctaccca cggagctcct gccatgcccc tga 873 41 1149 DNA Streptomyces lavendulae 41 atgaaattcg cttatttctc ccatgtctgg ggacgtcccg gtatcacgcc gggcgagcgc 60 tacgaagagc tgtggcgcga ggtcgaggac gccgaccggc tcggcttcga ctacgcgttc 120 tcggtggagc accactgcac gccgcacgag agctggatgc cctcgcccgc ggtcttctgc 180 acgggcgccg cgctgcgcac cgagcgcatc cgggtcggcc cgatggggtg ggtgccgccg 240 ctgcgccacc cgctgcacct ggtcgaggag gtcgcgaccc tggaccagct cctgggcggg 300 cggctggagg tggggctcgc ctcgggcgtc agccgtgacc ccttcctgcc cttcgacgcc 360 gatttcgaca accgtcacct cctgacccgg gaggccctgg agctgctgcg tgccgcgttc 420 gccgcgcggg gcgccttcga cttcgacggg cccgcgcacc ggctgcgcga catcgccctg 480 tccttcccgc cggtgcagcg cccgcacccg ccgatgtggg tgcccaccac caaccgcaac 540 accttgcgct atctcagcga ggccggtgcc cacaccagtt ccacgatgat cgtgccgcgc 600 gcctccatgg cgctggtcta ccggcactac ctcgactggt ggcgcggcca cggccacgcg 660 agcgacccgc gcatcggcta ctggacgctg gtccacgtgg cccggacgga cgccgaggcg 720 gaggagcggg cggccgcgca catcaccgag acgttcacca agacgctgcg gtacggctcg 780 gtgtcccgtt cccgcgatca gcacgcccca cccagcaggc tcagcacgac ggacatcctg 840 gcgggctccg gcgacctgcg cttcctgctg gagaacaacc tcgtcttcgt cggctcgccg 900 gcgaccgtgg ccgaccggat cagggccgcg tccctggagg gccatttcga cacgctgctg 960 ggcgagttca ccttcggcga gctggcggac cggcaccgca tcgagtccat ggaactgttc 1020 gcgcacgagg tggccccggc actgcgcgcc ttctccccct acgcgccgcg cccgcaggag 1080 ccggcgtaca ccgcgagcga cgagcagcag gtggcggccc gcctccaggc tctgggctac 1140 atcgactga 1149 42 1215 DNA Streptomyces lavendulae 42 gtgacaccgc cgacgacagc tcgcgaaccc ctccggatgg cagtgctggg cgcgggatgg 60 gtctcgcgca aggtgtggct gccgctgctg gcggaacacc cggcgttccg ggtcgacttc 120 ctcgtggacg acgaccccgt ggcggccagg tcggccctgc cggagggcgc gcggacccgc 180 gtcctgagca gaccggaaga gctcgccccc agaagcgtgg acgcggccat catcgccctg 240 cccaaccacc tccatctccc cgtggccaag gccctcctgg agcgggacgt gccggtgttc 300 gtcgagaagc cggtgtgccg cacgctcttc gaggcccagg cgctcgccct ggaccaccag 360 gcgcggggcg acagcatcgg ggacatcacc ctctacgcct ggagcgccgc ccggcaccgc 420 accgatgtct gccgcctggc ggagctgctg ccctcgctgg gcaccgtgcg cagtgtcggg 480 ctgagctgga tccgggccac cggcatcccg cagcgcaccg ggtggttcgt cgaccgccgg 540 ctcgccgggg gcggcgcgct gctcgacctg ggctggcacc tgctggacgt gggcctgcac 600 ctgctggggt ggccgcgcgt ggtccgggcg gcgagcacga tgtccgcgga ctggatgagc 660 cggggcgagg ccacggccga ctggagccgg cgctcctccg gcacggcgcg gccaggcccc 720 ggggagacgg tggaggacac cgcccgcggc ttcctcgtca ccgacaccga cgtgggcatc 780 tccctggaga cacgctgggc ctcccaccag gcgctggacg tcaccacgat caccgtggag 840 ggcaccgagg gggtggcgac gctgcgcggc accttcggct tcagccccca ccggctacag 900 aagtcgagcc tcgtggtcct gcgccagggg gtggaggaga ccgtcgcgct gcccgacgag 960 cccgtgggcg tggagtaccg gcggcaggtg gacgaactcg cccgcaggct cggcggctcg 1020 gccgacgggc agggcccggt gtcgggcctg ggcgaggggt cgatggccga agtgaccatc 1080 ctggcctcct gcatcgacca catctattcg gccgccggcg tcgaccctcc ctcgcccctg 1140 caccggccgc agagcgacgc ggcgcccagc acgtccagtt gtccacgtgt cctgcccacc 1200 cggggaagcc aatga 1215 43 774 DNA Streptomyces lavendulae 43 atgagcaccg tcaccgaccg ggccacggag cgcctgggac agagcggccg cgtggtcgtg 60 gtctcgggcg cgtccgggca gataggcggc gcgtgcgcgc tggagctcgc cgcgctcggc 120 gccaccgtcg tcgccggcta ccacagcggc gagcaggcga tccgcaagct gcgggagcag 180 gtggagggcc agggcggcac cctcgtgccc gtggcggcgg acctgagcga acccgagggc 240 gccgacgcgc tggtggcggc ggccgtcgaa cggttcgggc gggtggacgg ctgtgtggct 300 gctgcgggct tgcgtacgcg ccggctcgcg atggccacgg acgcccggag cctggagaag 360 ctgctgcggg tcaacctggc cggttccgtg ggtctcgcca aggcgtgcct gaagccgatg 420 atgcgcgcca ggtacgggcg gatcgtgctc ttcggctccc gggccgggac cagcgggctg 480 cccggccaca gcgcgtacgc cgccaccaag ggggcgctcc agccgtgggc ggcgtcggtg 540 gcgggtgagg tcggcaagca cggcatcacc gtcaacgtcg tcgcgcccgg ggcgatccgc 600 gccgaggtga tggacttctc ggaggccgag cgcgatctgg tcctgcagtt catcggggcg 660 gggcggctcg gtgagccgga ggaggtcgcg gcggcggtgt cgttcctgct gtcgccgtcg 720 gcctcgtacg tcaacggcaa tacgctcgtc gtcgacggtg gtgcccgctt ctga 774 44 2124 DNA Streptomyces lavendulae 44 gtggccctgc gcgcgcccaa cagcccgcgg tgggtcgtcg ccttcctctc cctgctggca 60 tccggcgcca ggcccctgct gctcgaaccc gacacccccg gccccgagac cgcgcggctg 120 ctgcgggctg ccgggggcgg caggtccctg gtcgtccccg ggaccggcga cggcctccgc 180 ctgacgttga ccggctcgcc cggagaaccc tccggcgccc cgcccgccgt gctgctcccg 240 acctcggggt cgaccggtgc gagcaagctc gtcgcccgca gcgaggagag cctgctcgcg 300 gagggccgcc gctaccgcga cggggtcggg ctgacgggag aggacaccct gctgctgccg 360 gtgccgctgt cccacgcgta cgcgctgggc tggctgttcg gcggactgct gacgggtgcc 420 gcgctgcgcc ccgtaccgcc gaccgccctc ggccgcatcg ccgcggagct gtccggtggt 480 gcgaccgtgg tggccctggt gcccagtgtg gcccggctgc tggcgacccg gcggctgcgg 540 ggagcagcgg ccgggcgggc gcccgccgct cccggtctcc ggctggccat ggtgggtgcg 600 gggccggtgg acgagcagct ggaccgcgcg ttcaccgagg cgttcgggac cggtctcgcc 660 cgcaactacg gttccacgga gacgggcgcc gtgctcgccg gaccggcggg gctggagccc 720 ttgtgcgccg gtgctcccct gccgggggtg gagtgcgaac tgaccggccc ggagggcgtg 780 gtgccgcccg ccggcacccc ggggctgctg agcgtacggg tcgacggccg gccgtacgcc 840 atgggcgatc tcgccgtggc cgtgcccggg ggcctgcgca tcctgggacg cgaggaccgg 900 gcgatccgcc ggggcgggcg ctgggtctcc ccgctggaga tcgaggaggt gctgcgcggt 960 catccggacg tggtgaatgt gcgggtgggc gcccggcggg ggcggcaccg gggcgaggac 1020 gggatcgtcg cggaggtctc ggcggcgggg ccggggctca cccccgaggc gctgcgcgag 1080 cacgcccgcc gggagctggc cccgcacaag gtgcccgacg agttcgtcct gcgggagagc 1140 ctgccggtca acgccgcggg caaggtgcgg gcggcgtccg tctaccgcct cacccggagc 1200 gcggcggagg ccgcccgggc gtacaaggca tccgaagtgc tcttcgcgct gcacgacttg 1260 ggcgccctgg aggcactcgc ccagggtgcc ggcacggctc tcctcgccgg ggagctgggg 1320 tgcgacgcgg atgccctgga gtggctgctg cgcacggcca ccgctctggg ggtgctgacc 1380 accggggcgc aagagcccgg ggaccgggtc cgggccgggg agctggccgc gttcgtggcg 1440 ctggaggagc acctctcccg tgggctggtc acgcgcgagg agctcgtcgc ggtggcccgg 1500 agcgggacgg cgcggcgtcc cttcgaggag cgtccccccg agagcctcgg tccgctcgtc 1560 gccctgtacc agggcgcgat ggacggcccc ggcgcacggg cccgggccgc gctcggcctg 1620 cggctcctgc ggcccgggcc gggagcccgg gtggtggagg tgaccgcggg cccgggccgc 1680 tatctggaac gcctgctcgc ctcggacccc ggggcgagcg gccatctggt caccgtcggc 1740 cggctgagcg ggccgctctc ctcggccgtc gccgcggcgg tcgaggaggg cagggtgacc 1800 gtggggacgg aactgcccgt cggctacgcc gacttctgcg tggtcgccaa cgccgtgcac 1860 ggcccggggc cgggcagcgc tctcggtgcc ctgctcggct ccctgcggcc gggcgggcgg 1920 ctgctggtcg acgacgtctt cctgccggcg tccgggccgg ggagcgaact ggctctggac 1980 tggctcacgc acggcgggac cgcgtggccg gccaccggcg agctgatcgc cgggctgctg 2040 caagaggggg cggaggtcgc acggcacgtg ccgctggacg cgtccccctg tcatctgatc 2100 atcgccaagg aggccggttc atga 2124 45 1152 DNA Streptomyces lavendulae 45 atgtcccgta gcacccaccc gccgacagcc acccccgacg cgggcaccag gcgacgcctg 60 ccgctgatcg gcaacgacct ggtcatcaac gaggactcct gcaacctcag ctgcacctac 120 tgcctcaccg gacagagcaa cctcaaggag ggccactccc ttcaactgat cttcgagccc 180 ccgcggcgcg acagctacgc caaggacagc gggctggggc agcgcatgga caaggtcgcc 240 gaccggatcc gggaccgctt cggcctgccg ctgctcaagg tgaccggagg cgagatcttc 300 ctggtccggg ggatcatgga cttcctggag caggaggccc gtaaatacga cgtgctggtc 360 atccagacca acggtgtcct ggtgcgcgag gagcacctgg agcggttccg ctcgtggggc 420 aacgtcgtgc tccaggtctc cctcgacagc cacctccacc acggcaacag ccatcgtgtg 480 ccgtccggga gcctgcacga gaaggtcgtc gccgccatcg cccggatcct ggactcgggg 540 ctgccggtgg agatctattc agtgctcaac gaccggagcg tcacggaggt ctgcgcgttc 600 gccgagtggc tgtcgggatt ctcccggcct cccgtctact tccccttccc ggtgcggggc 660 ccggactcgg aggacttcaa ggtgcggccc gggcagttcg gccacatcca ggaactcgtc 720 gaccgctacg acgagttcgc gcgggtcctc ccgccgcggc cctacttcga ccggctgacg 780 agcttctacc gcgagggccg ccgcaccttc cgctgccatc tgccgcggct ggtcgtctcc 840 agcttcagcg acggcgtcgt cacgccctgc cccaacatct ggttctccga catgggcaac 900 gccctggagg acgactggag cgagatgctg gacacggtgg gcaccagcgg cctctaccgt 960 gccctgctcg cccccaagcc ccggctcaag gcgtgccacg gctgcttcac gccctgggac 1020 acgctctcga tgtacttcga ggacgagatc accctcgacg agctgtgcgc cgctcccacc 1080 tactccccgc cccgcatccg gcagatgctc agcgacgcga aggccgacta cctccagggc 1140 ggccatgact ga 1152 46 21 DNA Streptomyces lavendulae 46 ggcaaggcat gcgagggtcg c 21 47 30 DNA Artificial Sequence A primer 47 ttccagaacg gcgccctgat gaccgccggc 30 48 30 DNA Artificial Sequence A primer 48 gccggcggtc atcagggcgc cgttctggaa 30 49 1545 DNA Streptomyces lavendulae 49 gtgccaccct ctccccgcgc cctcgtcatc ggaatcgacg gaggcacatt cgatacggtc 60 gacccgctga tcgagtgcgg tctgctgccc catatggcga agttgctgcg cgagagcgcc 120 agtgccgcca cggactgcac ctggcccgcc cacacggcgc cggggtggag cacgttcgtc 180 tccgccagcg atcccggcgg tcacgggatc tatcagttct acgacaccca ggacccggcc 240 tacggggccc gcgtcacgcg ctccggcgac ctgggccggt cctgcgcctg ggactggctc 300 gccgcgcagg aatattcgct gggcctcatc aacatcccga tgtcgcaccc gccggccgac 360 ctccccggct atcaggtcac ctggccgctg gagcggacac tcaagcactg ccgcccggat 420 tccctgctgc gcgaactcgc cgcggccaag gcccatttcc agtcggacct cgcgaccatg 480 ttccggggcg acatggccta tctggaggag gccgagcgca atgtggcggc gcgggtccgc 540 tccgtacggc atctgatgag cacccggccc accgatgtcg tgatggtcgt gctcaccgag 600 gccgaccggg tcggccacca ctactggcac tacggcgacc ccggtcaccc gggccaccgg 660 cccgccccgg agggcagcgg ctgggacgtc gccatgcccc ggatctacca ggccatcgac 720 cacgcggtgg gcgagctcct ggagctcgtg gacgaggaca cctccgtcgt gctcgtctcc 780 gaccacggcc tgggcaccgg gcgccacggc ctgtcggtgc acaccctcct ggaggaggcc 840 gggctgctgg ccaccgcacc gggggaggag ccgcaggacg cggcggcgag ctggttcgcg 900 ggcaacggcc ggcacgtcga cttccgccgc accagcgtct acatgcccgt ccccggcagc 960 tacggcctca acatcaacgt acgcggacgc cagcagcgcg gcaccgtcgc accccgcgac 1020 cgcgaacgcg tcatggacga ggtcacgggc ctgctctccg ggctgaccgg ccccgaggga 1080 cagcaggtct tccgggccgt ccgcccgcgc gaagaggcgt acccagggcc gcacaccggc 1140 cgggcacccg acctcctcct cgtcccgcgg gacgagaccg tcctgcccgt ccccgacctc 1200 ggcggtgacg tgtggcggcc gagcgcgcag accggcctgc accgctaccg cggcctgtgg 1260 gcgcaccgct cgccccgcgt ccgccccggc cgcctgcccg gcaccgtcgc gctcaccgac 1320 accctgccca ccctgctcac cgacctcggg gccgcatggc ccagcgacat ccacggccgc 1380 cccgtgaccg ccgtcctcga cgacggcgta cgcgtcccgc cctccgaccc ccgggtcgag 1440 gccaccggca ccccggccac cacgatcccg gccgccgctt cggccgctga tgccgccgag 1500 gacgcgtaca ccagcgaccg cttgcgcgaa atgggctacc tgtaa 1545 50 282 DNA Streptomyces lavendulae 50 atggagaccc tgacgaccga caagatcaag gaccggctgc gcaaggtgct cgtcgattcc 60 ctcgaactgt ccctggaccc ctcggccgta cccgacgagg gactcgtgga gaagctgggc 120 ctggactcga tcaacaccat cgaattcctc atctgggtcg agagcgaatt cggcatagag 180 atcgccgacg aggacctgtc gatcaagctc atcgacagtc tcgacctcct cgccggctat 240 gtgtccgagc gcgtgaacgg cgtcaccgca cccgccgaat ga 282 51 1413 DNA Streptomyces lavendulae 51 atggaccggc acgccctggt gatcgggctc gacggcatgc cgaggaccct gctgacccgc 60 ctggccggcg acgggaccat gccgcacacc gcggcgctgc tcgccgaggg ccactgcgcg 120 gaactgctgg cacccgtacc ggagatcagc tccacctcct gggccacctt cctcaccggc 180 accaacccgg gccggcacgg catctacggc ttcaccgacc tcgcccccgg cgacggctac 240 cgcatcacct tccccggtgt gcggcagctg cgcgaacccc cgctgtggga actcgccgcc 300 cgcgccggcc gcaggaccgt gtgcctgaac gtgccgggca cctaccccgc ccccgccatc 360 gacggcgtgc tggtctccgg cttcgtcgcg cccgaactgg agcgcgccgt cagcccgcca 420 cggctgctgc cgctgctgcg cggcctcgac tacgaactcg acgtcgaggt cggcgacgtc 480 gccgccgacc cggccgcctt cctcgggcgg gccgtccggg ccctgcgcgc ccgcacccgg 540 gcgatggaac acctgctgcg ccaggagacc tgggacctcg cggtcgccgt gctcaccgag 600 accgaccgcg tccaccactt cctgtggcgc gcggtcgccg accccgccga ccccctccac 660 ggggacgtcc tcgccttcta ccgcctcgtg gacgactgcg tcgccaccct ggtgagcacc 720 ctcccaccgg gcggcgaact cttcctgatg agcgaccacg gcttcggacc cgccgcctgt 780 caggtctatc tgaacgcgtg gctcagggag tccggctggc tggccgggct cgacgtctgt 840 ccggacctca ccgcggtcga cgctcgcagc accgccttcg cgctcgaccc cgcccgcatc 900 cacctcaacc gcaagagccg cttccccggc ggcggcctga ccgacgcgga ggcggacgag 960 gccgcccacg agatcgcgcg cgagctgtcc gccctgcgct gcgacggcac ccgcctgggc 1020 cccgacgtcg acggacccct gctcgtccgc gacctctacc gcgctcagga gatctaccac 1080 ggcccgctgt tgggcaacgc ccccgacctg gtggccgtac cggcccccgg ggtgcagctg 1140 cgcggcggct ggggcggcac gcacaccgta cgcaacgaca tcctcaccgg cacccacacc 1200 cgcgacgacg cggtcttcta ccggcgcggc gcgcccgcgc ccgcccccgg ggcggacgac 1260 ggccccctcg acatgacgga cgtcgccccg accgtcctcg cctccctggg catccacccc 1320 ggcgggctcg acggcgcggc cgtactcggc accacgggac ccgcgtccgg tcacggccgc 1380 acggaccccc ctctcgacat cagggagctc tga 1413 52 1836 DNA Streptomyces lavendulae 52 atgaagcacg acctcggtct ggcaccatcg gcacccaaac cgggaacact cgacctgagc 60 ctggacccac gcatcacgga ccccgcttcc ttccgggtca gtttcctgat cctcctcgac 120 ggcgacctcg tgatgtcccc cgaacacctc ggcgtcgcct acatggccgg tgtgctgcgc 180 catacgggct tcaccgcgga gatccgggag gtggagcacg gcgacgacca ggcggccgcc 240 accgtcgagg cgctcaagga gtaccggccc gacctcgtct gcttcaccct gatgagcctg 300 aacctgggca gctgtctgac cctgtgccgg atgctgcggg aggagctgcc ggggacgacg 360 atcgcctgcg gcggcccagc cgggaccttc gcgggcctgg acgtcctgcg gaacaacccc 420 tggaccgacg tcgtcgccgt gggggagggc gagcccacca tcctcgacct cgtccaacgg 480 ctctacctca aggagccgtt gtccgcctgc aaggggatct gctaccgcga cgaggacggc 540 acaccgcgcc agaaccccgc ccgccccctg atccacaacc tggaggacct ccccttcccc 600 gcccgggacc agctgcgcca gcacggcgac aagctggagt acgtccgggt cagcaccagc 660 cggggctgcg tcgccaactg cgccttctgc tccgccccgc acctgaagaa ccgcgtccag 720 gcgggcaagg cgtggcgcgg ccgcgggccg gaacagatcg tggacgaggt cgccgagatc 780 gtcgaacgcc accagttccg gaccttcgac ttcgtcgact ccaccttcga ggaccccgac 840 ggcggccggg tcggcaagaa acgggtcgcc gccatcgcga acggcatcct ggagcgcggc 900 ctcgacatct actacaacgt ctgcatgcgg gccgagaact ggcacgacac ccccgaggac 960 cacgccctgc tcgacctgct ggtcgcctcg ggcctggaga aggtcaacgt cggcatcgag 1020 gccggcaccg ccgaggaact gctcctctgg gagaagcgcg ccaccgtcga ggacaacgtc 1080 accatcatca ggatgctgcg ggaacacggc atctatctcg ccatgggatt cattcccttc 1140 cacccctacg cgaccctgga gaccatcgtc accaacgcgg ccttcctgcg cgacaattcc 1200 ggccacaacc tccggcgcat gaccgaacgc ctggagatct accccggaac ggccatcgtc 1260 agccgcatgc gggccgacgg actcctcggc gagagctatc tcgaagggct cgacccctac 1320 ggctacgcat tcaaggatcc ccgcgtcgga cggctcgcca agcatttcgc ccagctctac 1380 aacaacgacg actaccaccg gcacggcgtc atcaccgagc agtcctccgt cttcgccttc 1440 gagacctaca acgtcgtact ccagaccttc atctcccggc tgcaccgccg gttcaccacc 1500 ctgccggggg tggacgaggt gatggaggca ttcaaggccc gggtgcacga gatccgccag 1560 gagatgggcc ggcacaacta cggcttcttc atgtccaatg tcgaggcggt catgaacgac 1620 accctcgacc cggagaagca gcgccggcag gtggtggacg tcgagcactt cttccgcgac 1680 cgcctcgatg tgttgcgcag cgagcaattg cgcgtcggca aggccctcac ccggctcggc 1740 gcccgggtga cggaggtcag ctcgaccatt cccaaggagc gccccggcgg actgccgcgc 1800 cagtacacgg gagagggcag cggtgccacg tggtga 1836 53 1080 DNA Streptomyces lavendulae 53 gtgccacgtg gtgagacggg aaccgccgcg gcgcgggtgg cggtctgcac gctgagcagc 60 agggaactgg tcggcccgct ggcccggttg cccggtgtgg cggccgcggg cacgctgatg 120 accgccaacc tgggcatcga gcaggtgatc aaggccctgc ggtgcgaccg gacggtccgc 180 ggcctgctcg tgtgcggccg cgactcaccc cgcttccgcg ccggccagag cctgatcgcc 240 ctcttccgcc acggcctgcg ccccgaggac gggcacatcc ggggagccac cggctatctc 300 cccgtcctga ggtcggtgac ggcgcgggag accgaggagg tacgcgcccg cgtcgagctg 360 gtggacgccc gtggcgagcg cgacgtcgag acgctgcgcg ccgaggtcgc ggcactcctc 420 gcccgcgtac ggcgcacccc ggccctcccc tcccgcgagc acgacggcgg ccaacccagc 480 ttcgtggagc cggacttcgg acggctgcat cctgtcggcc gccgccgctc cctggacgcg 540 ggcatcggcg ggttcgtgct catcagcgtc gaccgtgagc accggcggat cctgctgcgc 600 cactacacct ccgatgtgcg gccccggcac gagatgtggg gcacccgcgg ggaggcgatg 660 ctgctcgggc tgctggaggc cggcgtcatc gaggaccccg cccacgccgg atacctcggc 720 gccgaactgg ccaaggccga gacggcgctg cggctcggcc tgcactacga acaggacctg 780 cccctgcgcc cgccgggcag gccgcccggc cctgtgcggc gccggaccgc gaaggagcga 840 acgaccatgg cgcaagcacc cgcgctggag gacttcctgc gtctcgtgac gaggacgctg 900 ggggccgagg acgccgtcct ggacctgcac acgccgctcg gcgagcaact ggcggtggac 960 tccgcccggc tcatcgaact caccgtcgtc ctggaggagg agctcggcgc ggacctcccc 1020 gacgacgccg acctcgccag ggccaccccc gcggaactcc acaaagcact cgtgggctga 1080 54 438 DNA Streptomyces lavendulae 54 atgcgcagcg tgctgttgct caacggaccc aacctgggga cgctcggcaa gcggcaaccg 60 gagatctacg gaaccgacac cctggccgag atcgaggccg ccgtggccga ggaggtggga 120 gcgcgcggct gggaggtggt ctccgaacag cgcaacggcg agggggaact ggtcgatgtg 180 ctccagcgcc acgacgacgt ggtgggcgcc gtggtcaacc ccggcgccct gatgatcgcc 240 ggctggtcac tgcgcgacgc gctcgccgac ttcgccccgc cctgggtgga ggtgcacctg 300 agcaacgtgt ggggacgcga ggcattccgg cacacctccg tcacggcccc gctggcctcc 360 ggcgtcgtga tggggatggg ggcgctgggc taccggctgg cagcgcgcgc cctcacccgg 420 ctggtccccg aggactga 438 55 534 DNA Streptomyces lavendulae 55 gtgggacggt acggaagaga gggtctgggg atgtcgcgta cggctgaggg gaacgccgga 60 ggcgtggtgg tgccggtggt ccggctggtc gccgtgacgg acgggccgga cgcggagggc 120 tggcggcagg cgctcgcccc cgaactggtg gtggagcacg gcgtcgaggc gatcgcggag 180 gcggccgggg acggcgggcc gtgggcgctg gtctgtgccg gtgccgggct gggcgcggcg 240 ctgcgggccg ccgagcgggc cgcgcgcccg ccggtgcatg tgctgctgtg gctcggcagc 300 cgcgggcccg gcgaaggggt gggcggggag gtctccggtc aatttccctg tccggtcacg 360 gccttggtgt ccgcggaggt ggaccgcggt cgcgccgtgg tccccgcctg gcgcggcctg 420 accgaggggc cgttcaccgt gcggatcctc ccggcggcct gcccgctgcc cggggcgtgc 480 gaccaggccg gcgctcaggt gatcaaggag gagctgcggg tgtggcccgc ctga 534 56 765 DNA Streptomyces lavendulae 56 atggatgcga ctttgacgaa tgacgtcgag aaagcctccc gggatctggt cgaagccgga 60 tactgcctga tcgagtgccc cttgccggcc gcggtcttcg aaaagctcag agggcggctg 120 ctggaggtcg ccgagcagga gcgtgagaac ggctcggcct ttctctacga cggcggcaac 180 caacgcgtct tcagcctgct gaacaagggc gaggaattcg agcagaacgt gcaggatccc 240 accgtcatgc tcctgatgga ggagatcctg ggcttcggct tcctgctctc cagcacgcac 300 gccaatatcg cgggccccgg cggttcccgg atgcatctgc acgcggacca gaccttcgcc 360 cgcccgccgt ggcccccgta tccgctggtg gccaacagca tgtggatgct ggacgacttc 420 accgaggaca acggcgcgac ccgcctggtg cccggctccc atctgctggg ccggcagccg 480 gactacgacc ggggcgaggg gaacaccgag acggtcgccg tgtgcgcgcc ggccgggagc 540 gtgatggtct tcgacgggcg cctgtggcac cagacgggcg ccaacaccac cgaccggccg 600 cggcacggca tcctcaacta ctactgccgc ggctacgtcc ggcagcagca gaacttcttc 660 tcgggtctgc gggaggacgt cgccacccgc gcgacgcccg aactgcgccg gctgctgggg 720 tacgagaact acttctccct cgggatgacc gacggcctgc cgtag 765 57 795 DNA Streptomyces lavendulae 57 atggcacact caccgcggcg gccggacggc cccctccgca tcggggtctg gctggccccc 60 cagcacacct cggtggccga actgcgcgcc gcctggcgcg cggccgactc cctgggcgtg 120 gactcgctgt ggctgtggga ccacttcttc ccgctcaccg gggaccccga cggcagccac 180 ttcgaggcct ggaccctgct ggcggccatg gccgccgaca cccgcgccgc ccgcctgggc 240 accctggtgt ccaactacgc ctaccgcaac cccgacctcc tggccgacat ggcccgcacg 300 gtcgaccaca tcggcgacgg ccgcctgatc ctcggcatgg gcgccggctg ggtcgaacgc 360 gacctgaagg agtacggcta ccccacgccc ggcgcggggg agcgggtgga cgggctcatc 420 gaggcggtgg agcgcgtcga ccgcagactc ggccggctgc gccccgggcc gctcggcgac 480 ctccccctgc tcatcggcgg ggacgggcag cggcgcctgc tgcgcttcgc cgccgaacgg 540 gccgccatct ggaacaccat ggcctggcgc ttcgccgagg gcaatcgcgt gctggacgag 600 tggtgcgcgc gggtcggccg cgacccggcg gagatcgagc gcagcgcctt cgtcacccgc 660 gaccagaccg acgaggagct gcgctgcctg gtggcgacgg gcgtccagca cctgatcttc 720 caggtcgggc accccttccg cttcgacggc gtggagcggg ccctgcgctt cgcgggcggc 780 tggagcaagg ggtaa 795 58 825 DNA Streptomyces lavendulae 58 atgaagatca gcattgctct gccgaacacc gtgcccggcg cggacgggcg cctgataacc 60 gattgggcgc ggcgggccga ggagcgggga ttcgcctcgc tcgcggccac cgagcgcctg 120 gtgtatccgg gccacgatcc gctgctggcg ctggcggcgg cggccggggc gacctcccgg 180 atcgggctgc tcaccaatgt cctgatcggc ccgctgcgca ccgcgcctgt gctggcgaag 240 gcggtcgcga gtctggactc gctgtcgggc gggcggttca ccctgggggt cgggcccggc 300 gtgcgcgagg acgacttcga ggccgccggc cgcgccttcg acgaccggcg cgcggcgttc 360 gaggagcagc tggagctgct cggccggggc gcccggccgg gcgcggaggg ccccggtgtg 420 ccggtcctcg tcggcggggt cagcgcggcg gccgtgcgcc gcgtggcgcg ctgggccgac 480 ggctggacgg cgcccggcct ggagccggag cggatcgtgc cggtcgcgga acgggtgcgc 540 cgcgcctgga gcgaggcggg acgcgccggg gcgccgcatg tggtggcgct ggcgcgctac 600 accctgggcg aggacgtggc ccaggagtcg gcggccttcg tccgggacta cttcgcggtg 660 ctgggcgagg aggcggagga gttcgtggcg aagaccccgc gcaccgcggg gcagctccgc 720 gcggcggtct cggcgctcgc cgacggcggg gtggacgagg tcgtcctcca ccccacggcg 780 gcggcgctgt cccaagtgga ccggctggcg gacgcgttgc tctag 825 59 1383 DNA Streptomyces lavendulae 59 atgcccgctg ccggaaaagt cgccgtgata ggactcgact ccgcgactcc gcagtacatg 60 ttcgaccggt tcgccgagga catgccggtg ttcaccgccc tcaggcgcaa gtccctgtgg 120 ggtccgatgc gcagcatcga cccgcccatc accatgcccg cctggtcctg catgatgtcc 180 ggccgctcgc ccggcgaact cggcgtctac ggattccgcg accgcggcgc ctacgactac 240 gggccgttga agttcgccac ctcccacagc atccaagccc cccggatctg ggacgagatg 300 acggccgccg ggcgctccag cgtggtcctg ggcgtccccg gcacctatcc tcccgccccc 360 atccgcgggg ccatggtctc ctgcttcctg gctccctcca cacagtcgcg ctacacctcc 420 ccgcccggcc tcgccgacga gctggagaag ctcaccggcg gctacgccct ggacgtggag 480 gacttccgct ccaccgacct ggaacgcgta tcccagcgcg tcttcgacat gagcgagcag 540 cgcttcgagg tcgcgcgcca cctggcgacc acccaggagt gggacttcct ctccttcgtg 600 gacatgggcc ccgaccgcct ccaccacggc ttctggaaat actgcgaccc cgaccacccg 660 cgccacgagc cgggcaacgc ctacgccggt ctcttccgcg actactaccg cgccctcgac 720 cggcacctcg gccgcttcct ggagagcctg cccgagaaca cgaccgtcct ggtcgtctcc 780 gaccacggcg cccagccgat ggtgggcggg ctcttcgtca acgagtggct gcgcaaggag 840 ggttacctcg tcctgaccga ggagcccgcc ggacccaccc ccgtcgccca ggccgccgtc 900 gactggaagc ggaccaccgc ctgggccgaa ggcggctact acggacggat cttcctcaac 960 gtcgagggcc gggagccgca gggcaccatc ccggccgcgg agtacgagag cacccgcgac 1020 ctcatcgcct ccgccctgga agcgctgccc gacgaccagg ggcagccgat gggcacccgc 1080 gccctgcgcc ccggcgagct ctacggagag gtcaacggca tcgcccccga cctcctggtc 1140 tacgtcggca acctgcgctg gcgggccctg gccaccctcg gcatgggcaa gggcctctac 1200 acgacggaga acgacaccgg ccctgaccac gccaaccacg gggacaccgg catcttcgcc 1260 ctcagcgccc ccggcatcac ccccggccgc gcggacggcc tgtcgctgta cgacgtggcc 1320 cccaccctgc gggaactgct gggtctcgcg ccgcagggct cccgcggctc cctcctcggc 1380 tga 1383 60 1536 DNA Streptomyces lavendulae 60 gtgaaggcga tggaccgggt ggacagagcg gtcgagcggt tcccgatgta catcgacggg 60 caggccgtgc aggcccacga cggcgccgtc ctgcgcacct tcgagccggc cacgcggcgc 120 cacctggccg accttcccag cggcggcgcg gaggacgtcc gccgggcggt gtccgccgcc 180 cggcgggcct tcgacgaggg cccgtggccg cggatggcgc cgggcgagcg ggcgggcctg 240 ctgcgcaagg ccgcacagcg cttgcgtgaa gaagcggagc cgctggccga gttggaggcc 300 cgcgacaacg gctcgacgct gcgcaaggct ctcggggccg atgtgccggg ggccgcggca 360 gccttcgagt ggagcgcgtg gtgggcggag cacgtgcccg aacggcagcc ggaggcgccc 420 ggttcgggtt cctacgtcgt gtggcggccg gtgggggtcg tcgccgcgat cgtgccgtgg 480 aatctgccgc tgctgctggc ggcctggcgc atcgcgcccg ccatcgccgc gggcaacacc 540 tgtgtgatca aaccggcttc gttcgcctcg ctctccacgc tgcgactggt ggagctgctc 600 cacgagtgcg gcctgccgcc gggcgtggtc aacgtggtca cggggccggg cggggtcgcc 660 ggggagcagc tggtgcgctc gcccggcgtc gacctggtgg cgttcaccgg ctcggacgag 720 accggggccg ccgtacggga gggtgccgcc gcggcgggga cgagcgcccg gctggacctg 780 gggggcaagt cccccaacat cgtgctcgcg gacgccgatc tggaccgggc ggtcaccggc 840 gtcacgtggg gagcgttcct gcacaacggg caggtgtgca tggccggtac ccgcgcggtg 900 gtgcacgccg acgtccacga cgacttcctg cggctgctga gcgaacgggt gggccggctg 960 cgcgtcggtg atccgctgga cccggccacc gacctggggc cgctggtctc gcgcaaccag 1020 gcgcgtacgg ccaggcgctt caccgaactc gggctctccc agggcgcgga gctcgtgtgc 1080 ggcggccggg cgcccgcggc ggacgagctg ccgcccgggc tggacgccgg ggcgtatttc 1140 ctgcccacgg tgctggcgtc ggtcggcgcg gacgacgccg tcgcgcagga ggagatcttc 1200 ggcccggtgc tcgcggtcgt ccgggccggg tccgacgacg acgcggtgcg catcgccaac 1260 ggctcccgct accggctcag cgccggggtg tggtccgccg atcccgcgcg ggcccgcgcg 1320 gtggccgagc ggctgcgcgc ggaccgggta tggatcaacg actaccggct ggtcgacctg 1380 gagctgcccg gcacagccgg gccccgctcc gccgtctggg accggctcac caacgagctg 1440 gacgcctacc gccacaagca cgtggtgcac ggtggcggtg cgggagcggg cggggtgccg 1500 gcgccgccca ctccctacgc gctgctgggc gggtga 1536 61 1419 DNA Streptomyces lavendulae 61 gtgaaaccag ccagccactc cgtgacggac acgtccgcgg ccctcggcgc cgcggccgcc 60 gaagagctcg cggcgcaggt cgccggatcc gtcctcctgc ccggggacga ggggtacgac 120 gaggagcgct ccggcttcga actgtccgtg gaacaccgcc ccgccctcgt cgtcgtcgcc 180 accggtgccg cggatgtcat cgccgccgtg cgcttcgcca gggcccgggg ccttgggatc 240 gccgtccagg ccaccggtca cgggaagtcc tcggcggcca ccgacgtcct catcagcacc 300 cggcggatga ccggcgtcag ggtcgacccg cgggcccgga ccgcccggat cgaggcgggc 360 gtgcgctggg agcaggtgat ccacgaggcg gcggcgcacg gtcttgcacc gctgagcggc 420 tcggcgccgt tcgtcggcgc ggtctcctac ctcctcggcg gcgggctcgg gcttctgtcg 480 cggaagtacg ggttcgccgg cgaccatgtc gtctcgctcg acctggtgac ggccgacggg 540 cggtttctcc aggtctccgc cgaggaacac cccgatctct tctggggcgt gcgcggcagc 600 agggggaacc tcggcatcgt cacctccgtc gaggtcgggc tgttccccgt cacccaggtg 660 tacggcggag ggctgttctt cgacgccggc tccacgcgcg ccgtgctgaa cacctatctc 720 cagtgggcgc cccggatgcc cgaggacatg gcgtcgtcgg tgttcctggc cgcgtatccc 780 gatgccgagg gggtgcccgg accgctgcgc ggccggttcg tcacccacat ccggctggcc 840 tggctgggag accccgagga gggtgagcgc cggttcgccg agctccgggc cgccggcacg 900 gtcgtcatgg atacggtgga cacgctcccg tacacgcggg ccgggatcat ccacaacgat 960 ccgccggccc cggtgtcgag tcacagcaaa acggtcatgt tcgggcagct ggacgagatc 1020 gccgtcgacg agatcctcag gctcgcgggg ccgggcacgg acgcgctgtt cggggtggag 1080 ctgcggcacc tgggcggcgc cctcgcccgg ccgccccggc acccgagcgc ggtgggccac 1140 ttcccggagg cggtgttcaa cgcctacgtg ggctcgctgg tcgacccgga caccctggcg 1200 gccgtggacg cggcgcagca ggagttcgtc gacagcatgc ggccgtggac gacgcccggg 1260 gtgtgcctga acttcctcgc gggtcacaac acatcgaggg agacgacccg cagcgcctac 1320 acgccggagg actacgcgcg gctccaggcc ctgaagtcgc agtacgaccc gggcaacgtc 1380 ttccggttca accccaacat cccgcccctg ccggcctga 1419 62 1188 DNA Streptomyces lavendulae 62 atgacctcag ccgccccgcc cgcctttccc ttcccgcccg gccccggcgg cacggtgccg 60 cccgagtacg cgcggctgct caccgatgac ccggtcgccg aggtgcgcct ggcggacggc 120 tcgcgcatct ggctggtgac ccggcacgag gacgtgcgca cggtgctcac cgacggccgc 180 ttcagccgcc atcgcgccgc catgctgccg ggctcgggct tcggccggtc ccagggctcg 240 ggcatcgtgg acctcgaccc gccggagcac ggccggctgc gcggtccggt ggtggccgcg 300 ttcggtgcct cgcgcacggc gcggttcgca ccccgcatcg aggcggccgc cgaggcggcc 360 ctggaccggc tgcccgccgg cagcggcacg gtggacctcg tcgcggcgta caccgcgccc 420 ttcgccggcc gcgtcacagc cgagttcctc gggctgcccg gggaccggtg gcaggacgtc 480 acctccgacg tcgagctgct gctgcttccg cgcggtgcca ccgagcaggc gctgaaggag 540 gcccgcggca ggctcggcca ggtgctggac gaactgctcg cggcccgcag ggccgagccg 600 ggcgacagcg tcaccgacac gctgctggac gcggaggagc tcaccgacga cgaccggcgc 660 ctgctgctcc acggcctgat catctcgggc ttcatcacca tccgcgacct gctggcccgg 720 cacctcttcg gcgtgctctc ctcccccggc ctcgcggccc ggctgcgcga ggacccctcc 780 gtactgccct ctgccgtaca ggagttgctg cgctactacc cctccagcaa cgacggcctg 840 ctgcgggtcg ccaccgagga cgtggtgctc tccggcaggc gcgtcgccgc cggggacgcc 900 gtgctgccac tggtctcggc ggcctcccgc gaccccgagg tcttcgccga tccccacgtg 960 ctcgacatcg agcgggtggc cgaccgcggc atcgcgttcg gcgccgggca gcacgcctgc 1020 cccgcgaccg ggctggccgt gaccgaactg accgtcggca tcggccgcct gctggcggcc 1080 ttcccccgca tcgccctggc cgtgcctccc gaagaggtcg agcacagctc cgaactcctg 1140 cccctgggcg tccggtcact gccggtggtc cccggcccgc gcaactga 1188 63 1425 DNA Streptomyces lavendulae 63 atgcttcctg agttccaatt gcagtggaat tggctcgacg ccccggccgg cggcggaggc 60 gagctgcaag cgacctgggc ccggctgcgc atcgccgtgg gcgccgagac cgtcacactc 120 gtccaggagc ccgggcaggg gaccttccgg gagcacacga ccggctcgct ctaccccctg 180 gccgagtgga tcgccttcaa ctggtggtcg ctggtggccg acgcgcggcc cggcacccag 240 atatcccagc tgcgcttcgc ctaccgccac ggtgtgggcg acaaccgcgg ttcgtggtgg 300 atgcgttcgc gccgtcacat cctgcgcgcc gcctgcgacg gcttccgctg gccggacatg 360 ctcttcgtgc ccgagggccg ggagacccgg atcgtatgga tgccggacat gggccccgac 420 gtacgacccg ggaaccgctt cgcgagccgg ggcaactcct gtgtggagag cgccgcgttc 480 accgccacac tggcctcgtt cgtcgacgcg gtgaccgagc gcctcacgga ccagggcatc 540 accggcaccc cgctccagga ggagtgggcc gccgtccgcg ccaccgacga ggacgaggcc 600 gccttctgcc gcatcgcggc acggctgggc ctggacccct acgccgaggc cgagccgtac 660 gaggcggaca tcctcaaggc cgccgagcag ttggcggaac cgctcgccag tgacttcttc 720 aacggggtgc ggcctgagcg gatagccgac cagctccagt ggatcgcgcg cgtccgcacc 780 ctgatgggca ccgcgcccgc ggataccccg ctccctcccg ccttggtgga actgcgcaag 840 gactgcgccg acttgagcga gaagttcttc gctccggggc gactcgacaa cccctgggac 900 ctcggctacg aggtggcgca ccgggtgcgc gcgtgggcgg gtctggacga caccgcgccc 960 ttcgacccgg cccccctgat gggctaccgc accgagcagg tcccctatat ggaccggggc 1020 ctggtcgccc tcggcacccg caggggcgcg gacgggccgg tcctggtctc ctcccggcgc 1080 ttcaccgacc gcccgcgccg cttcctccag gcccgcgcgc tgtggcatct gatctgcgac 1140 cccgacgaca ccttcctgat cgcggcggcg cacacccacc gccagcacgt ggcccgcggc 1200 ttcgccctgg aggtcctggc ccccgccaag ggcgtggcga ccctgctggc cgaccccgga 1260 cacctggtgt ccgccgagga cgtcgaggtc atcgccgacg actacggctg cggcaacatc 1320 gtcgtggaac accagctgga caaccgcgtc ctggcgaagg acttcacctg gccgggccac 1380 gcccgcgccg gcgcgccggc cggtgagagg agccggggcg catga 1425 64 1332 DNA Streptomyces lavendulae 64 gtgacaatcc gccagcgtgt cgtcgtcgtc atcaccgagg gagcggcacc cgagctgctc 60 gaccgctggt gtgcccaggg gctgctgccc ggcttcgccg ccctgcgctc gcagggggct 120 tccgggccgc tccacgccga gggcaccccc tacgaaccgc cgggcctgct gagcgtcctg 180 accggccggc gcgccgcgga ccacggcttc tactcctact ggacctgtca cgacccggag 240 tacgcgccgc aggtcctcac ccccgagcac cgccgccacc cactgctgtg gcagcacgag 300 gtgttccagg gcgtcaggtt cgcctcgata ggcctcttcg gcacccatcc cccggagccc 360 ttcgacggtt ccctgatcac ctatccgatg tatgccaccc tccacgcctg ccacccgcgc 420 agcctccagc gcaccctggc gaagaagggc atccgtccgg tccacgacgt gtcgatcttc 480 tggaccgggc aggaccgcga cgagctgctg ccttccctgc tggaggcgga cgtgcagcgc 540 gggcgcgcgg cattggctct gctggaggag tccgatgtcg tgatcgtcaa cctcacgagc 600 atcgaccgct gttcgcacat ctactggcag gagctggagc acggccccga gcacgagcgg 660 gagagcgccg tcttcgccgc ctaccgcacc tgcgaccagg tcatccagga cgccctgcgg 720 gcggccgacg accgcaccag tgtcgtggcc ttctcggaga taggcttcgg gccgctgcgc 780 aactactgtt ccatcaacga cgagatggag caggcgggtt tcctggccac cgccgaggac 840 ggccgcgtcg agtgggccgg cagcgcggcc ttcgaggcgg tgcagggcac gcacggggtg 900 aacatcaacc tgcgcgaccg ctacaagcac ggcctggtcc cggagcgcga ctacgagaag 960 gtccgcaccg acgtcgcggc cgcgctgctg gagcggcgca acccccgtac cggcaggctg 1020 ttcttcgacg cggtgcgccg ccgggaggag gtctatcccg gcgaggccac ccagcacgcc 1080 cccgacctca tcctggagcc ggcggactgg cgctatcttc cgctgggcga cccgcactgg 1140 gcctcgcacg tccaccgcga ctggcagagc ggctggcacc gccgggagtc ctactggtcg 1200 gccgtcggcc ccggcttcac cggtggggcg cggcagaccc gcaccgccgc ccccgtcgat 1260 attcccgcga ccgtatgcgc tctgctcggg cgtgacgtgc cgaacgactg ggacggcgtg 1320 ccgctgtcct ga 1332 65 372 DNA Streptomyces lavendulae 65 atgacaccag aggaactctc cgacttcgcg ctggagctgc cggaggcggt ggacgacgag 60 gcgttcggcc ccggagccgc ggtcttcaag gtggagaaga aggtcttcgc cattctccag 120 gacgcctccg aggaccgccc gccgcaggtc acgctgaagt gcgaaccgga tctggcgctg 180 cacctgcgcg agcagtacgc ggcggtggtg cccggctacc acgtcaacaa gcgccactgg 240 aacacggttg tcctgaacgg cacggttccc gtggaggagc tgcgggagat ggtggagcat 300 tcgtacgatc gcgtggtggc ggggctgccc aaggcggtac gggaacgtct gcgcctcctg 360 cgcaccgtgt ga 372 66 1056 DNA Streptomyces lavendulae 66 atgaccgtgg agcagacccc cgagaatccc gggaccgcgg cccgcgccgc cgcggaagag 60 accgtgaacg acatcctgca aggggcgtgg aaggcccgcg ccatccacgt ggccgtcgaa 120 ctcggcgtcc cggaactgct ccaggagggc ccccgcaccg cgaccgccct cgccgaggcc 180 accggcgccc acgagcagac cctgcgcaga ctgctccgac tgctcgccac ggtgggcgtc 240 ttcgacgacc tcggccacga cgacctgttc gcccagaacg ccctctccgc cgtcctgctg 300 cccgaccccg cgagcccggt cgccaccgac gcgcgcttcc aggcggcccc ctggcactgg 360 cgggcctggg aacagctcac gcacagcgtc cgcaccggtg aggcgtcctt tccttcgacg 420 tggccaacgg cacctcgttc tggcagctca cccacgaggg accccaaggc gcgcgaactg 480 ttcaaccgcg ccatggggtc ggtctccctc accgaggccg gacaggtcgc cgcggcctac 540 gacttctccg gcgccgcgac cgccgtggac atcggcggcg gccgcggcag cctcatggcg 600 gccgtcctcg acgccttccc cggcctgcgc ggaaccctgc tggagcgccc gcccgtcgcc 660 gaggaggccc gtgagctcct caccggccgc ggcctcgcgg accggtgcga gatcctgccc 720 ggcgacttct tcgagaccat ccccgacggc gccgacgtct acctcatcaa gcacgtgctg 780 cacgactggg acgacgacga cgtcgtacgc atcctccgcc ggatcgccac cgccatgaag 840 ccggactccc ggctcctggt catcgacaac ctcatcgacg agcggcccgc cgcatcgacg 900 ctcttcgtcg acctgctgct gctcgtcctc gtcggcggcg ccgaacgctc ggagagcgaa 960 ttcgccgcgc tgctggagaa gtcgggcctg agggtggagc gctcgctgcc ctgcggcgcc 1020 ggcccggtgc gcatcgtcga gatccgcagg gcctga 1056 67 1641 DNA Streptomyces lavendulae 67 atgacggtgc tgggtctggg tggatccgga catgactggg cctcctgtgc caccgacggc 60 cgacggctgg tggcgatcga cgaggagcgg ctggtccgca gcaagtacgg cctgggagcg 120 gacctcctgg cgggccacag ccggcgcgcc gtcctcgacg ccctcggcac gagtgccgag 180 gccgtggaac acgtggtggc ctgcgagctc gtaccacgcc ccttctacca ctcgttccgc 240 aggcgcgtga cggtcgtcaa ccaccatctc gcccacgcct acagcgcgtt cggggcctcc 300 gggatgaccc gcgccgccgt actggtctgc gacaactccg gcagcctggt gacgggcctg 360 aagtccggcc cagggccgcg cgaggcggag acgatcagct gctacaccgc cgacgcctcc 420 gggctgcgcc tggtcaaccg ggtcgccggg acacacgccg tggacgcctc ctccgagagc 480 gcctactacc agcccggcga gaccgacaat tccctcggcc acttctaccg ctcggccagc 540 ctcgcactcg gcctcgccta ctccggtccc aagacccgct accccgtcag cgaggacggc 600 aagaccatgg gcctcgcgcc ctacggcgac gaccgcttcg tcgacgaggt cgcggagctg 660 gtcaccctgc tgcccgaggg cggcgtgcag atctcggcga gcaaggtgaa ccacctcttc 720 gaacgcctcg tggaatcggg tgagttcgag gaccgggcgg ccttggccta cgccgcccag 780 gagacgctgg aacgcgccct gctgcactgc gcccgcgacc tgcaccgccg caccggcctg 840 acggacctgt gcatcgccgg cggcgtcggc ctcaacagcg tcgccaacgg ccggatcctg 900 cgcgagaccc ccttcgagcg ggtcttcgtc gtcccggccg cgggcgacaa cgggatcagc 960 ctcggctgcg cctactacgg cctccacgag ctggaggggc gcgcgccgtc ggagctcccc 1020 gccctcgaca ccgcctacct cgggcccgac taccccgccg agcgcgtcga cgcggcgctg 1080 gccggctcgg gcttcaccgt ggagaccccc gacgacctgc ccggcagggt cgccggcctg 1140 ctcgccgaag ggaagatcat cggctggttc gacggccgct ccgaattcgg cccgcgcgca 1200 ctgggacacc gcagcatcct cgccgcaccc ttccccgcct ccgtgcggga ccacctcaac 1260 gacaacgtca aacaccgcga gtggttccgc ccctacgccc ccatcgtccg cgaggaccgg 1320 gcggcggact acttcgacct cgtccagccc tccccgttca tgctggtcgt cgcgcgcgtg 1380 acccggcagg acgccatccc cgccgccacc cacgtggacg gcaccgcccg gctccagacg 1440 ctgaacgccg cacagaaccc gaaggtctac gagctgctcg gcaggttcga ggcgctcacc 1500 ggctgcgccg tgctgctcaa cacctccttc aacgtcgccg gccagcccat cgtcgagacc 1560 ccggaggacg ccgtcgaggc gttcgcgggc atgcgcctgg accacctcgt cgtgggggac 1620 cggctggcga ccaagccctg a 1641 68 1707 DNA Streptomyces lavendulae 68 gtggacgtcc ccgtgctcgt ggtcggagga ggaccgacgg gcttggcgat ggcgctcttc 60 ctcgcacgcc acggcgtcgg ctgcctgctg gtcgaacggc ggacgaccac ctcgcccgtc 120 ccgcgcgcca cccacgtcag ccgccgctcc atggaactct tccgcgaggc gggcctggag 180 gaggagatcc gccgggccgg gttcgaggtc gtgcgcgagg acgacccacg gctgcggacc 240 cggcccgaac gccacctgcc ccgggtggtc ctgcaagccg cctcgctcgc cggccccggc 300 ccggtggggg tcctggagac cggtgacgag gaactggccg tacccggccc ctgcgcaccc 360 ttctggtgcg gccaggaccg gatggaaccc ctgctcgcca aggccgcggc gcgccacggc 420 gccgatgtgc gcttcggcca cgaactgacc ggcctgtggc cgggggagga cagcacacgg 480 gcccgcgtcc gggcagcggg aacgggacgg acctacaccg tcgacgcccg cttcgtcatc 540 gccgccgacg gggcgcgcgg cgagatcgcc gagcgcgtgg gcatcgcgcg ggagggcctg 600 ggcacggtcg cccaccgggt gagcatcctc ttccgcgccg acccggggcg ctgggcccgc 660 gaccggcggt tcttcatgtg catgatccag aacccggggt tcgacggggc ggtgatggag 720 ctcaacaccc cgggccgctg gtgcgccgcg gtggactacg acccggcccg cgccgaaccc 780 gacggcacct actccgcacg cacctgcctc gacctggtcc gggccgccgt cggtgacgac 840 cggagcgacg cggcggtcga caccgtcttc cactggaagg cccggcaccg catagcggcc 900 gcctaccgca gtggggcggt gttcctcatc ggcgacgccg cccacctcca cccgccctcc 960 ggcggctacg gatccaacgt cggcttccag gacgcgcaca acctcgcctg gaagatcgcc 1020 gccgtgctcg gcggctgggc cggaccgcgg ctgctggaca cctacgacga agagcgccgc 1080 cccgtgggaa aggcgacggc ggagcagtcg atgctcctcg acggcgtgcc accggaacca 1140 ctggggggaa gcgtcgtccg ctgcgatccc cgcaccctga tcatgggata ccgctaccac 1200 tccgccgccg tcctcggccc cccgcacggc cccgccttcc ccgcggcctt caccctgcgc 1260 ggagacccgg gcacccggct gccgcacgta tggctgcgta cggacgcggg ggaacgcgtc 1320 tccacgctcg acctgtgcca cgggcacttc gtcctgctct ccgccgaccc ggtctgggcg 1380 gcggccgcgg cgcgctcggc gaaggagacg ggcgtaccgc tgcggggcca ccacctggcg 1440 gccaccggaa gcgaactcgc cgacccctcc ggcgagttcc cgcggagctg cgggaccggg 1500 cccgcggggg ccgtgctcgt acggccggac ggcatggtcg cctggcgcac ggcccgcgcc 1560 gtgcccccgg acccggacag cgcgcaggac ctggtcacgg cagcggtgag acgtgtcctc 1620 gcactgccgg agcgcgcggc gccaccggtg ctcggtccgc cgcggttgtc acgcggttcc 1680 tatcggcgag tcgggagcga cgggtga 1707 69 483 DNA Streptomyces lavendulae 69 gtgaagcctc attccttctg cacgtgctgg ccgggcgcca ccgtatggct gacgggccca 60 ccgggcgcgg gcaagacgac gatcgcccgc gcactggcgg agcggctgcg cgaacggggc 120 cggcgcgtgg aggtgctcga cggcgacgcg acccgcgcgc tcctgaccgc gggctcctcg 180 tgggaggacc gtggcaccgg cctccagcgg gtcggcctga tggccgaggt cctggcgcgc 240 aacggcatcg tcgtcctcgt cccggtgacc gcggcccgcg cggacagccg cgaagccgta 300 cgcagacgcc acgagcggtc cggcaccgcg cacctggaag tgcgggtggt ccgggacgca 360 gtgcctccga gcgggctccc cgcgccgccc ggcccagatc tgcggatcgc ggcgcacgag 420 cagagcgccg aggagtcggc gcgggcactg caccggctcc tggcggagag ggagctggcg 480 tga 483 70 960 DNA Streptomyces lavendulae 70 gtgaaccccg ggcgcggtgg agcgtacgcc gcggggcgcg acgggacccg cgggacgcga 60 cgccctcacg gtctgtcgca cctggatctg ctggagtcgg agtcggtcca catcttccgt 120 gaggtggcgg gcgagttcga gcggccggtg atcctcttct ccggcggcaa ggactcgatc 180 gtcatgctgc acctggcgct gaagtccttc gctcccgcac ccgtgccgtt cgcgctgctg 240 cacgtggaca ccggccacaa cttccccgag gtgatcgcct accgggaccg cgtcgtggcg 300 gcgctcggtc tgcggctgga agtggcctcc gtgcaggact tcatcgacaa cggcaccttg 360 cgcgaacgcc cggacggcac ccgcaatccg ctgcagacgg tgccactgct ggacgcgatc 420 gggcgccacc gcttcgacgc cgtcttcggc ggcggccgcc gcgacgagga gaaggcccgc 480 gcgaaggagc gggtgttctc cctgcgcgac gagttcggcg gctgggaccc gcgccgccag 540 cgccccgaac tgtggcggct ctacaacggc cgccacgcac ccggcgagca cgtccgcgtc 600 ttccccctct ccaactggac cgagctcgac gtgtggcagt acgtcgcccg cgaggagatc 660 gaactcccca ccatctacta cgcccacgag cgcgaggtct tccgccgcgg cggcatgtgg 720 ctggcaccgg gggagtgggg cggcccacgc gagggggaag cggtggagaa gcgacgggtg 780 cgctaccgca cggtggggga catgtcctgc accggcgcgg tggactcggc ggcggccacc 840 gtggccgacg tcgtcgccga gatcgccacg tcccgcctca cggaacgggg cgcgacccgg 900 gccgacgaca agctgtcgga agccgcgatg gaggaccgca agcgcgaggg gtatttctag 960 71 492 DNA Streptomyces lavendulae 71 gtggggcagg acagccggcc gcggtggctc accgacgagg aacaacgcgt gtggcgcggc 60 tatctgcggg ccaccaggct ggtggaggac cacctggacc gccgcctcca gcgggaagcg 120 gacatgccgc acctctatta cggtcttctc gtccagctct ccgaggcccc gcgccggggg 180 atccggatga ccgaccttgc ccgcaacgcg aagatcaccc gcccgcggct ctcgcacgcg 240 atcacccgcc tggagaagct cggctgggtg cgccgggaat cgtgccacgg cgacaggcgc 300 ggccagaacg ccgtcctcac ggaagagggc cgcgaggttc tggagaagtc ggcgccgggc 360 catgtcgccg ctgtgcgcgc ggccgtcttc gacagcctca ccccggaaca ggtcgggcaa 420 ctgggccgga tctgccaggc gatagagaag gggctggacc gggaaggcgc ggacctgccg 480 tggctgcgct ga 492 72 1242 DNA Streptomyces lavendulae 72 gtggaacgac acgacggggc accgggctgg ggcttcaccc atacccagta cagcgcggac 60 cacggtgaac gcggcgccac ccgcagggcc ggggccctgc tctccgcgcg gcccctgccg 120 cagaaccagc acatcatggg ctggggcgcg gagaatcccg aaccggcgcc cggacgctac 180 gacttcgagg tcctcgacga gcgcgtcgcc ctgatgcgcg cgacgggggc cacgcccgtc 240 ctgaccctgt gtgccgcccc cgactggatg aagggcggcc ggcccggccg caccgactgg 300 tcgcgactgg agaccgcccc cgacccccgg cactacgcgg acttcgcccg gctcgcgggc 360 gtgatcgccc aacgctaccc ggacatcagg cacttcctcg tgtggaacga gctgaagggc 420 ttctacgacg aggacaggcg gcgctgggat tatgagggat acacccggct gtacaacctc 480 gtccacgccg agctgaagcg gcggaacccg cgcaatctgg tgggcggccc ctatgcggtg 540 gtcgaccacg acccgcccgc cgaggacgcg gcggaccgct cgcgcgaact gcgcggtccc 600 tggggcgagc tggaccagcg ctccgccgac gtcatccgct attggaacgc ccacaaggcg 660 ggcgcggact tcgtcgtcgt cgacgggtcc agctacaccc gcgagggcca ccgggcgatt 720 ccggacgagt tcgccgccac cgagaagttc gccgacgtca cccgctgggt caggagcgtg 780 accggactcc cggtgtggtg ggccgagtgg tacgtcgagc cgcccgccga ggacgaccgg 840 ccgggcggcc gggacggctg gggcgagggg caccgcaccg ccgtgcaggc caccgcgatg 900 atgcggctgg cggagagcgg cgcgtcggcc gccttctact ggaacccgca gcggaccggg 960 aaggcgtgcc ccggctgcct gtggcggagc acccacttgc gcgacggggg aggggagttg 1020 cccatggcgg gtctcctgag ccggttcgct cgcgaattcc ctccgggcac cgccttccgg 1080 ccggtcgccg tcacctgcgg gagcggtgac agggtcgagg ccctcgccga cgaggccgcc 1140 gtgctcgtcg tcaacaccga gtgccggccg gtggccgcca gggtggacgg gcaggcgctg 1200 tccctcgcgc cgtacgaggt gcgctggctg acccgcccgt aa 1242 73 816 DNA Streptomyces lavendulae 73 atgacgcgaa ggcgcccaac gggcccgatt caccgtcggc gggcgtcact caccctttcc 60 cccacgggag ccgccatgag aagaaatcgc atcgccgccc tgctgccggc cgctctggca 120 ctggtcggca tatccgtcct cgcccccgcc accacggcga gcgcggccgc accgcacggc 180 ggcacctcgc aggccgccgc attccccgtg agcgaggccc agttcaagca gatgttcccg 240 aagcggaacg cgttctatac gtacaagggc ctggtcgccg cgctcaaggc gtacccgggc 300 ttcgcgggca ccggcagcgc cgaggtccgg aagcaggagg ccgccgcctt cctcgccaac 360 gtcgcccacg agaccggcgg actggtctat gtcgtggagc agaacaccgc caactacccc 420 cactactgcg accggagccg gccctacggc tgtccggcag gccaggccgc ctactacggt 480 cgcggcccgc tccagatcag ctggaacttc aactacaagg cggcgggtga cgccctcggc 540 atcgacctgc tccacaaccc ctcgctggtg cagaaggacg cggccgtctc ctggaagacc 600 ggcctgtggt actggaacac ccagcgcggc cccggcacca tgaccccgca cgaggccatg 660 gtcaaccacc gcggcttcgg gcagaccatc cgcagcatca acggcgccct ggagtgcgac 720 ggccacaacc ccgcccaggt ccagagccgc gtcgcgaact accagcgatt caccaagatc 780 ctcggcgtgg cgccgggcgg caatctctcc tgctga 816 74 12249 DNA Streptomyces lavendulae 74 ggatcccgat cgtctcggac atgaccggcg accttctcgg cgcgcgggag gcccaggacc 60 ccgcctactg ggtgtcccac atccgccgcg cggtgcgctt ccacgaccag atccgccgtc 120 tgcagcgcta cggggccggg gccttcgtcg aggtcggccc ggacacggtg ctcagctcgg 180 ccggccaggc gtgcctgacg gaccaggcgg gcaggagcgc gcccgtcctg gtgtccctcg 240 cgcacgccga gcgcgcggag gtgcccgcgc tcctgaccgc tctggccacc ctgcacaccc 300 gtggcgtggc cgtggactgg cgggcgtggt tcggcgacgg gccgcgcgcg gccggcctgc 360 ccacatacgc gttccagaag cagcactact ggccgtcggg ccccaccggt tggcggtccg 420 ggcccgcccc cgtacccctg ccccaggccg gaacggagga cgccgaaagg cccggtcgcg 480 ccgcggagtg gcgggcgctg ccgcccggtg agcggtacga cgcgctgctg cggatggtgc 540 gcggcgaagc cgccgccgtg atggggcacg ccgggccgga ggcggtggag ccggagcgcg 600 gcttcctcga ccacggcttc gactcggtga tggccgtgaa gctgcgcgac cgtctcgtgg 660 ccgggacggg gcgggagctg ccgacgaccc tgctgttcga ccaccccacg cccgcggccg 720 tcgccgacta cctgctggcg gggacgggcg aggccgagac ggcgccgtcc gtgtccctgt 780 cggaccagct cgaccgcctg gaggccgacc tcgcgcggct gccggccgac gaccggcagc 840 gcgcccgcgt cgccgagcgg ctcaagggcc tgctcgcggt ccacgcgccg gaccggggcg 900 ccgggagcga ggacgcgccg gaccaggacg cgctggacac ggcgaccgac gacgagatgt 960 tcgagctgat cgagaaggaa ctccgccgtg gatgagacca acgagaccaa actccgcgag 1020 tacctgcggc tggtcacggc cgatctgcgg cgaacccgca ggcagttgga ggaggccgag 1080 gacgcggccc gcgagcccgt cgcgatcgtg ggcatggcgt gccgcttccc cggggacgtg 1140 gcatcgccgg acgacctgtg gcagctggtc gccgagggcc gggacgccgt caccgagttc 1200 cccgccgacc ggggctggga cgtcgacgcc gtctacgacc ccgagccggg caccccgggc 1260 aggacgtacg cgcgccacgg cggcttcctc aaggacgccg ccggattcga cgccgccttc 1320 ttcggcatca cgccgcgcga ggcgctcgcc atggacccgc agcagcgcat gatcatggag 1380 gtctcctggg aggcgttcga gcaggcgggc ctcgacgcga ccaccctgcg gggcgaggac 1440 gtcggcgtct tcgtcggctc caacagcaac gactacctga tcaacgtgct cgacgcgcgg 1500 gacgtcgccg agggcttcat cgggaccggc aactccgcca gcatcctctc cggccgcgtc 1560 gcctacacct tcggcttcga gggcccggcc gtgtccgtcg acaccgcctg ctcctcctcg 1620 ctggtcgcgc tgcacctggc cgcgcagtcc ctgcggcagg gggagtgctc cctggcgctg 1680 gcgggcggcg cgacggtgat ggccacgccg accgccttca tcgagttcag ccgccagcgg 1740 ggcctggccc ccgacggccg ctgcaagtcc ttctcggcga ccgccgacgg caccacctgg 1800 tccgagggcg cggccgtgct gctgctggcc cggctctcgg acgcccgccg cctgggctac 1860 cccgtgcacg cggtcatccg gggcagcgcc gtcaaccagg acggcgcgag cgcgggcctg 1920 accgcgccca acggaccggc gcaacagcgg gtgatccggc aggcactggc caacgcacgg 1980 ctgacggccg acagcgtcga cgcggtcgag gcacacggca ccggcacccc gctgggcgac 2040 ccgatcgagg cccaggccct cctcgccacc tacgggcggg cccgcggcga gggcaggccg 2100 ctgtggctgg gctcgctgaa gtcgaacctg ggccacaccc agtccgcggc cggcgcgggc 2160 ggcgtcatca agatggtgat ggccatgcgg cacgggacgc tgccccgcac gctgcacctc 2220 acggagccca ccccgcgcgt cgactggtcc gccggtgacg tacggctgct gaccgaggcc 2280 caggactggc cggacaccgg acagccgcgc cgtgcggccg tctcgtcctt cggcgtcagc 2340 ggcaccaacg cccatgtgat cctggagggc ccgcccgccg aggaggcacc ggacgcgccg 2400 ctgccggacg tctcctcgca gccgcggggc ccgctgccgt gggtcgtctc cggccgcagc 2460 gaggcggccg tccgagcgca ggccgagcgc ctggcggccc acctgaccgc gcgcccgcac 2520 ctggcaccgg ccgacgtggc caccgcgctg gccaccacgc gggcggcctt cgaccaccgg 2580 gccgccgtcg tcggccggga ccgtgaggaa ctgctcgccg gcctcgcggc cctggccacc 2640 ggaacccgcg cgcccggcct ggtcaccggc cggaccccgc cgtccggcgg caaggccgcc 2700 ttcctcttca ccggacaggg cagccagcag cccggcatgg gccgcgaact ggcggctcac 2760 agcaccgtgt tcgccgacgc cctggacgag gtctgcgccc agctcgaccg gcacctcgac 2820 cggccgctgc gcgaggtgct gttcgccgcg gacggcacgc ccgaggccgc cctgctcgac 2880 acgacggcct acacccagcc cgcgctgttc gccgtcgagg tcgcgctgct gcggctgctg 2940 gaggactggg gcttgcggcc cggcatggtc gcgggccact cggtcggcga actgaccgcc 3000 gcctacgccg ccggggtctg gtcgctcgcc gacgcctgcg ccctggtcgc cgcccgcggc 3060 cggctgaccc aggcactgcc cgcgggcggc gccatggtcg ccgtgcaggc gaccgaggac 3120 gaggtgcgcg cccaactcgc cgacggccgc cccggcgtgg acatcgccgc cgtcaacgga 3180 ccggaagcgg tggtgctgtc cggcgacgag gccgccgtca cggacctggc gcgcgagtgg 3240 gccgcccgcg gccgggagac caggaggctg cgggtcagcc acgccttcca ctccgcccac 3300 ctggacgcca tgaccgaggc gttcgccgag gtcgcacgag gggtgtccta cagcgcgccg 3360 tccctcccgg tggtctccac gctcaccggg gcccccgtca ccgacgagct ccgcaggccg 3420 gaacactggg tgcggcacgt ccgggagacg gtgcgcttcc acgacgcggt ccgcgccctg 3480 cgcgaccgcg gggccaccgc gttcctggag gtcgggcccg gcggcgtgct gacggccgcg 3540 gcacgccgat gcctgcccga cgccgccccc gagacgttcg tccccgtgct gcggcgccgc 3600 aggcccgaac ccgagtccgt gctgacggcc gtcgcgcagg cccacacgat cggcctctcg 3660 ccggcgtggg accgcctgct gcccaaggcc cggacgcgcg tggacctgcc cacgtacgcc 3720 ttccagcgcg gccactactg gctggcgggc atggccggag cgggcaccgc gcggccggtg 3780 cggccggaag tgcaggagcc caccgccccc tccggtacgc cgccgctgtc gcgacggctg 3840 gccgacgcgt cggaggagga gcgcggccac ctgctgctga cgctggtacg cgagcagtcg 3900 gccaccgtga tgggcggcgt cgaccccgcg caggtcgaac ccgaccgccc cttcctggag 3960 ctcggcttcg actccctgat gggcgtcgag ctgcgcaccg cgctcgccgc cgactgcgca 4020 ctgcccctgc cgcccggcct gatcttcgac caccccacgc ccgccgccct ggccgccttc 4080 ctcggcgagc agctcgcggc ggcggcctcc ggcaccccca cggcggcggc accctcgccg 4140 tactccctgg aggcgctgta ccgcaacgcc aacaccctcg accggcccga ggacgcgctc 4200 gccctcacca aggccgcctc ccggctgcgc ccggtcttcg ccagcgtggc cgaggcgggg 4260 caggacccgg tcacggtgga gctggcacag gccaccggcc ttccgggcct gatctgctgc 4320 ccggcacccg tgccgctgta cggggcacag cagtacagcc ggctcgcagc cgccttccgc 4380 ggcacgcgcg gagtctcggc cctgctcgcc cccggcttct ccccgggcga actgctgccc 4440 gccgacttcg aggtgatgca ggacttcctc gccgaggggg tccggcggca gaccgacggc 4500 gcgcccttcg tcctcctggg ccactcctcc gggggctggt tcgcctacag cctggcggcc 4560 cacctggcgc gcaccgggcc gcgcccggag gccgtcgtgc tgctggacac ctatcagctg 4620 cacgacccgg cgctgcaccg catgcagcgc gaactcgccc agggcgtcct ggaccgcgag 4680 gaggacttcg gggcgatgac ggacgtacgg ctgagtgcca tgggcaaata cttcgacttc 4740 ttcaccgact gggtggccga ggacgccggt gtcccgacgc tgctgctgcg ggcctccgag 4800 cctctgggcg aggtcgtcga gggccaggag tggcgctcca cctggccgtt cgacagcacg 4860 gtcctcgaca cggaaggcga ccacttcgcc atggtcaacg accacgcgcc gcggacggcc 4920 caggccgtga acggctggct gtcgggcctc accggcggaa ggggctgagc gccggtggag 4980 acacgcaacg ccgaacggcc gtggatacgc agcttccacc ccgctcccca ggcccctgtg 5040 cggctgctgt gcctgccgca cgccgggggc tccgcgagcg cctacttcgc gctgtcgagg 5100 gaactggcgc cccgggtgga ggtgctcgcc gtgcagtacc ccgggcggca ggaccggcgc 5160 gacgagccgc tgctggactc gatcgaggcc ctgcgcgacg gggtcgccga ggccctgacg 5220 ccctggctgg accggccggt cgccctcttc ggccacagca tgggcgccgt ggtggcctac 5280 gagctggcgc ggctgctgtg ccaggacgcg ggcgtgccgc tcacccacct cttcgtctcc 5340 ggacgccggg gatccgaccg aagtctccgt ccttgccgcc gtgttccgga attcaccgtg 5400 acaccgccgc gcggctcttc ttccgaagtc ctccagatcc ggcacgagtt tgtatccgaa 5460 cggggttctg cgtgcgaaat actctcttcg aattgggtga catacccccg atcggcaccg 5520 tacccgagca gatgtacgcc tcggtgatcc gacgggagcg ctacggacag ccccaccagg 5580 cgttccgcag cgaggtcgtg gacgtgccga aggtggggcc cggtcaggcg ctggtcctcg 5640 tgatggccgc gggcatcaac tacaacaacg tctgggcctc cctggggcag ccggtcgacg 5700 tgatctccgc gcggcagaag cagggccaca gcgaggactt ccacatcggc gggtccgagg 5760 gctccggcgt ggtgtgggcg gtgggggagg gcgtcaccca ggtcgcggtg ggcgacgaag 5820 tgatcctctc cggctgccag tggacggaga cggccgccga catccggctc ggcgccgacc 5880 ccatgacctc cggctcgcag tcggtgtggg gatacgaggg caactacggc tccttcgccc 5940 agttcgccct cgtcgacgac tatcagtgcc accccaagcc gcccggcctg acctgggagg 6000 aagccgcctg cttcctgctc accggggcca ccgcctaccg ccagctgtgc ggctggcagc 6060 cgcacgacgt gcggccgggc gacccggtcc tcatctgggg cggggccggc gggctcggct 6120 ccatggccat ccagatcacc cgggcgcggg gcggcatccc cgtcgccgtg gtctccgacg 6180 aggagcgggc ccgctactgc cgggagctcg gcgcccaggg caccatcaac cgcctggact 6240 tcgaccactg gggacggctg cccgacatcg gcgaccacga ggcgatgggc cgctggaccg 6300 agggtgtacg ggccttcggc cggcgcttct gggaggtgct gggcgagcgc aggtccccgc 6360 gcatcgtcct ggagcacagc ggccaggcca ccatccccac ctcgatgtac ctgtgcgaca 6420 acgcgggcat ggtcgtcatc tgcggcggca ccaccggcta caacgccgac atcgacctgc 6480 gcttcctgtg gatgcgtcag aagcgcttgc agggctcgca cttcgccaac ctgcggcagt 6540 gccgcgacgt catccacatg gtcgcgaacg gccagctcga cccgtgcctg tcgtggaccg 6600 gcggcttcga cgacatcggc aaggcacacc agctgatgca cgacaaccag cacccccagg 6660 gcaaccaggc cgtcctggtc aacgcgccgc ggaccggcct gaccaccttc gcctgaacca 6720 ccgccccggt gttccgacgt cttcccccca cacttaccga ccaaggagag atcaccatgg 6780 acaagctcga catcctctgg agcgagcgcg agatccgtgc cgtgctgcag cgctactgcc 6840 gcgggctcga ccgcctcgac gaggaactgg tcaagtccgc ctaccacgag gacgcgcacg 6900 acgaccgcgg cgtcatccgc ggcaacgcac acgacttcgt caagcagatc gtcccgctcc 6960 tgcgcgacgc ctacaccggc accctgcaca ccctgcacgg cagcacgatc gagatcgacg 7020 gggatgccgc gggcgtggag tcctactgca ccgcctacca ctaccgcgag agcgacggca 7080 tcaagcgggt ggagcagttc gccgggcgct acgtcgaccg cttcgagcgg cgcgacggcg 7140 tctggaagat cgcccgccgg ctcgtgctga acgacttcag cctcgcccag gaggtgccgc 7200 tcgaccccgc cgaggcccag gccggcttca acccctccca ccgcgacctc accgacgcca 7260 gctaccaggt gctgccgctg cgcggcccgg acgcccccac cctctgagcc gtccggccgc 7320 cccaactcgc cccacctcac caggagtcac caccgtgtcc gacaccgagc agcacgcgcc 7380 cacgctgccg cggcagcgca cctgcccctt ctcgccgccg cccgagctcg aggagctgcg 7440 gcgcaccgat cccatcagca ggatgcggtt cgccgacgac tccccgggat ggctgctgac 7500 ccgccacgcc gacgtccgcg ccgcgctggc cgaccccggc gtcagctcgc accccggcaa 7560 ggcaccccag ccctggcgca acctcgcccc cgagatgcgc gccgagcact acctgccggg 7620 cttcctgatc ttcatggacc cgccggacca cacccgctac cgccgcctgc tcaccaagtg 7680 gttcaccatg cgggccatcc gcaagctcga acccaggatc gagcagatcg tcaccgagac 7740 cctcgacgcc atggaggccc agggcggcac cgtcgacctg gtgcagtcct tcgcgctgcc 7800 gatcccgctg ctggtcatct gcgagctgat gggcatccgc tacgaggagc gcgaggagtt 7860 catggacatg gtcctgcgac tccaggccct ggacgccacg cccgaggaac tcggggccct 7920 cggcgccagg atgaacgagt tcatgatgaa gctcgccgcc gccaagcgcg cgaaccccgg 7980 cgacgacctg ctcagccacc tcgcccacga ccccgacgcc gacccggcgc tcacggatct 8040 ggagatcgcc ggcatcggcg tgctgatgct catcgcgggg cacgagacct cggccaacat 8100 gctgggcgtc ggcacctaca ccctgctgga gaacgccgac cagtgggccc tgctccgtga 8160 cgacatcagc ctgatcgacc gggccgtcga ggagctgctg cgccaccaga ccatcgtcca 8220 gcagggcctg ccgcgcggcg tcacccggga catggagatc gccgggcacc aggtgaagac 8280 cggggagtcc ctgctggcct cgctgcccgc cgccaaccgc gaccccgccg tcttccccga 8340 ccccgaccgc ctcgacatca cgcgcgagca caacccgcac ctcgccttcg gccacggcat 8400 ccacctctgc ctgggcatgg agctcgcccg ggtggagatg cgccaggcgt ggcgcggcct 8460 cgtcacgcgc ttccccggcc tgcgcatggc cgccgcgccc gaggacatcc gctggcgcga 8520 cgaccagatc gtctacggcg tgtacaacct cccggtgacc tgggacgagg ccaagtgacc 8580 ggccccgagg ccgcggtgcg cgggtgcccc ttcggcgccg gcgaggcgcc cgcgtacccc 8640 ttccacgccc ccgaccggct ggagcccgac ccgtactggg agccgctgcg ccgcgagcgg 8700 ccgctgcaac gcgtcacgct gccgtacggc ggcgaggcgt ggctcgccac ccgctatcag 8760 gacgtgcgcg cggtcttcgc cgaccgcagg ttctcccggc agctcgccgt cgcgcccggc 8820 gctccgcgct tcctcccgca ccagccgccg ccggacgccg tcctgagcgt cgagggcccc 8880 gaccacgcgc ggctgcgccg gctggtcggg aaggtcttca cgccgcgccg cgtggaggac 8940 atgcgtccgc tcatccagcg caccgccgac ggactcctcg acgcgatgga ggagatgggg 9000 ccgcccgcgg acctggtcga ggacttctcc ctgcccttcg ccgtgtccat gatctgcgag 9060 ctgctcggcg tgccgcccga ggaccgcaag cggttctgcg tctggtcgga cgcgctgctg 9120 acgaccaccg cgcacacccc cgcccaggtg cgcgactaca tgatgcagat gcacgactac 9180 ctcggcgggc tcgtcgcgca gcgccgggtg cggcccaccg cggacctgat cggctccctc 9240 gtgaccgcgc gcgacgagga ggacaagctc accgagggcg agctggtgcg gctggccgag 9300 gccatcctca tcgccggcta cgagacctcg gcgagccaga tccccaactt cctctacgtc 9360 ctcttccgcc acccgcagct gctggagcgg atcaggaacg accacgacct catccccgac 9420 gccgtcgagg aactgctgcg cttcgtgccc atcggcaccg tggacggctt tccccgtacg 9480 gccaccgagg acgtcgagct cgggggagtc ctggtcaggg ccggggagac ggtcgtgccg 9540 tcgatgggcg ccgccaaccg cgaccccgag ctgttcacgg accccgacga gctggacctc 9600 gcgcggcggc cgaatccgca cctgggcttc ggcgcgggac cgcaccactg cctgggcgcc 9660 caactggccc gggtggagct ccagatcacg ctcacgacgc tgttccgcag atacccccgc 9720 ctgcggctgg ccgtgccgga ggagagcctc tcgtggaagg aggggctgat ggtccgcggc 9780 atgcacacca tgccggtcac ctggtgagga caccggcgtc ctcctgacct tcccggcgtt 9840 ctcacgccgt cccggcagcc ttccttccga cacgagcgca cagagggtga agcgaccgca 9900 atgagcacca tcgacgaatg ggaacacagc acgaaggagg cgggcatgga ccccgcggcc 9960 ctcagacgcc tgaccgatgt ggtgcgggcg aggggcggcg cggcgcagct gtgcgtcatg 10020 cggcggggca ccgtggtcct ggaccgctcg ttcggctgct cctccgactc cctcttcctc 10080 gtctacgcgg ccaccaagcc cgtcgccgcc ctcgccgtgc acgcgctcgc cgagcggggc 10140 ctgatcgggc tggaccggcc ggtggccgaa tactggccgc agttcgcccg gcacggcaag 10200 ggtgacgtga ccgtccgtca tgtcctccag caccgggccg gggtgccggt cggccggggc 10260 atcgtgcgca cgatgcgcac cgccggcgac tgggagcgct ccgtgcgcga ccttgagcag 10320 tcccggccca agtggcccgg cggcgaggtc gccgcctacc acttcatgag tttcggattc 10380 attctcggcg aactggtgca gcgcgtcacc gggcggtcgt tccgagattt cgtgacttcc 10440 gagctcttcg ccccacttgg gctgaatgat ttgcacatgg gattgcccgg cagtgcctgg 10500 ccccggcatg tgcccgcgcg ggccgcccac ccctccgaat ggcccaatca gtggatgagc 10560 aaccgccgcg gctaccgcca ggccgtcatt ccgtccgccg gtctttccgg aaccgccgca 10620 caaatggccc gcttttacca gatgcttatg gagggcggct cgctcgacgg catccgcgtg 10680 ctgcggcccg aaactgtgga ggaagccaga aaaccgtcca atgacggcgg aatcgacgct 10740 tccctcaagc gtccggtccg ctggtcccac ggattcatgc tcggtggtcc gggcccggac 10800 ccgcgggggc tgtccaatgt gctgggccgc acgagcgacc cgagcgcctt cgggcacgcg 10860 ggcaccacgt ccagcgtcgt gtgggccgac cccacgcgcg agctggtcct cgcctacctc 10920 tccaacatcc agcccgagtt cggagcgggt atcgagcggc tccgcgaggt cagtgacctc 10980 gcgctcggtg cctgcgaggc aggctgaccc gagccgtgcc gccacggccc ggcgcccgcc 11040 cgatccgatc gggtccggtg ggggccggcc gggtccgggc ggggacgcac ttcccccggc 11100 gtccccgccc gggccccggt gcgaaccggg cgcaaaggcg gccgatcgcc cggcgcggcc 11160 ggatgccccc gaacggtgtg aaacgttctt atcagcctct gaccagcacc gagtgatcta 11220 ctgcacagcc cgaggccgcg attccggcag tatcttgatc ttgacggggc accaatgcga 11280 gcgggctatt cgccgcggtt ttccctgacg tcggatgcag atgacaccgg aggagggcca 11340 gtgctgaatc tgcccaaagg aatggagcgc gcgcatccgc attctccgcc acaggtggga 11400 atactcggac ccttggaagt ccgctcggcc ggaggtgccg gaacgggagc cgcggtaagc 11460 ggtattcgcg tacgcacatt gcttgccgcg ttgactgccc gcctggggca ggcgatgtcg 11520 accgagcgca tcctcaaaga ggtctgggcc gacaacccgc ccgcgaccga tcgcaaggcg 11580 gtggccgtcg ccgtcctgcg gctgcggcgg gtcctcggcg acaacgaagg acggtggctg 11640 ctcacccgcc cctccggtta cgtcctggac atccccccgg accacctcga cgccgtacgc 11700 gcggagaccc tggtgcggga aggccgggcc gccctggccg ccggcgaccc acgcgtcgcg 11760 gcccgccacc tcacgcgcgc cctcgaccag tggcggggcg agccctacgc ggacgccaac 11820 gccatctcga ccgtgtccca gcgcatcacg gagctggaga acctcaggtc cgaggccgtc 11880 caggcgcaca tcgacgccag gctcgaactg ggtcaccacc aggaactggt cggcgaactc 11940 cgctcgctga ccgccgcgaa ccccctgcac gagccgcact ggctgcagct gatgctcgcc 12000 ctctaccgct ccggcaagca ggccgaggct ctcgccgcct atatgcagct gcggcaggcg 12060 ctggccgaga acctgggcat cgacccgggt cgtcagctcc aggaactgca cctgcggatc 12120 ctgcgcgccg acgcgggcct gctgacgggg tccgggccgg cggcaccggc cgagccactg 12180 ctcgtacggc agtcctgagg gctcacggcc acccgaagaa cgcgcggtag cacggaacct 12240 gctgctcca 12249 75 18034 DNA Streptomyces lavendulae unsure (302)...(302) n is a or t or g or c 75 cccaggacct cgtcgcggtc ccggccgcgt ggtggacctc cgccaacccc aacaacgacc 60 agctctgcca gggcatatcg gtggaggtca gctacaacgg caggaccatc agagtgccgg 120 tgcgggacaa gtgcccttcg tgcgaccgga cccacatcga cctcagcagg acggccttcc 180 agaagctggc gccgctcgac aggggtgtgg tcaacggcat cacctggaag ttcgtccgct 240 gacgccacgc cggggtcccc aaagcccggg accccggcgc tccgcgcccg gcacgccggg 300 gnccgcccgg cgtgtcggcg tgaggttcgt cgccttcaag agtcataaag acaatcgcga 360 ctgttgacgt tatgagttca tcaaatttaa ggtcgcggga ctcttggaac agatcaagac 420 gacggagaac aatgacgtac tcccccggcg cgcggccgcg cccggcccgg ctgtccgcac 480 tgctgctcgc aggcgcgctc gtcgcctcgg tgccgcccgc ggccgccgcg cgagcgccgc 540 aaccccccac cgccgaccgc ccccgcaccg ccgcctcccc cacaggcggc tgccgtacgg 600 gtgacggctg gacactcgac tccacccgca tcgaccccga cgacacccac cacgcctatg 660 tcggcaacgg ctacctgggg cagcgcgtac cgcccaacgg cgccggctac accgacagcg 720 acaccaagac cggctggccg ctcttcgctc cggcctacga cggctcgttc gtgtccgggc 780 tctacgcgca caacaagcag accgccgccg accggcaggt gatcgccgct ctgcccacct 840 ggaccggact ggccgtcggc accggcggcg agcacggcga tatcttcaac tcttcgacga 900 agtcgggccg gatttccgga tatcaccaga ccctcttcca gagctgcggc atcgtccgta 960 ccgccctgac ctggaccgcc gccgacggcc gcaggaccga cctggtctac gaggtgctgg 1020 ccgaccgcga cgacccgcac acgggcgccg tacggctgag catgacgccg cgctggagcg 1080 gcgaggccac cgtcaccgac cagctggacg gacgcggcgc gcggcgcatg cggcagaccg 1140 gcggcggcga ccgcaccggt gggaccggcc gggacggccg caccatggac gtggccttcc 1200 gcaccgacgg cacggacacc gacggcgccg tcgcctccac cctgagggcc gggcgcggtg 1260 tgcacacgac cggggaccga cgcgccgcgg ccgcgaagga cttgagcgtg aaccagtccc 1320 tcacgttccc cgtccgtgcg ggccacgcgt acgaactcac caaatacgtg ggtgtcgaca 1380 ccgcgctcac ctcgcacgcg ccccgcgagg acgccaccac cgcctccctg cgcgccgccc 1440 gccgcggctg ggacgggctg ctgcgtgccc acaccgccgc ctgggcccgg ctgtggcgct 1500 ccgacatcga gctgccggga cagcgcgacc tccaggcgtg ggtgcgttcc gcccagtacg 1560 ggctgctgtc cagcacccgg cagggggcat ccaacagcat cgccccggcc gggctgacca 1620 gcgacaacta cgcgggcctg gtgttctggg acgccgagac ctggatgtac ccggccctgc 1680 tggccaccgc gccccaactc gccaggaccg tcgtcgacta ccgctaccgc accctcgccg 1740 gagcgcgcga gaacgcccac aagctcggct accaagggct cttctacccc tggaacagcg 1800 gcagcgaggg cgacctggcc caggagtgcc acagcgtcga cccgccccac tgccgcaccc 1860 agatccacct ccagtcggac atctccctcg ccacctggca gttctacctc gccaccggcg 1920 acaccgcctg gctgcgcgag cgcggctggc cggtgatgga gggcatcgcc gaattctggg 1980 ccgggcgggt cacccccaac gccgacggca gctactccat caaggacacc gccggccccg 2040 acgaatacag caacggcgtc gacgacgcgg tcttcaccaa cgccggtgcc gccaccgccc 2100 tgcgcgacgc cgcccgtgcc gcgcggctgc tgggcgagcg cgccccggcg gagtggacga 2160 cgatcgccga ccggatccgc atcccgtacg acgcgcggca caaggtcttc gagcagtacg 2220 acggctaccc gggcagcaag atcaagcagg ccgacacggt gctgctgatg taccccctgg 2280 agtggccgat gtcccaggcc gacgcggcgc gcaccctcga ctactacgcc cggcgcaccg 2340 accccgacgg ccccgccatg acggactcgg tccacgccat cgacgccgcg gccacgggcg 2400 agccgggctg ctcggcgtac acctatctcc agcgttccgt ccggcccttc gtgcgcggtc 2460 ctttcgacca gttctcggaa gcccgcggca ccaaggccgg cgccgacgac cccctggccg 2520 gctcgcccgc ccacgacttc ctcaccggca agggcggctt cctccagatc ttcaccaacg 2580 gcctgaccgg catgcggatg cgcgaggacc ggctgcacct cgacccgatg ctgcccccgc 2640 agctcggccg cggcgtcacc ctgcgcggcc tgcactggca gggccgcacg tacgacatcg 2700 ccatcggcgc ccacgagacc accgtgcggc tcaccggggg tgcgcccatg accctctaca 2760 ccccgcaggg cgagcacgtg ctgaccaagg cggcaccggc cgtgctcaag acccgccgcc 2820 ccgacctcgc tcccaccgac aacgtggccc gctgcaccac cgccggtgcc tcctccgagg 2880 aacccggtat gtacgcggca gccgcggtcg acggcaaccc cgccaccgcc tgggtccccg 2940 acgggccgaa cggtgaactg accaccgacc tcggcaagtc cgtacgcgtc accaaggcca 3000 cccccgtctg gagcggcccg gcaccggcct cgtacagcgt ccagctctcc ctcgacggcc 3060 ggcactggca cgacgcggtc gcgggcggcg ctccggtgtc cgcgcggtac gtacgcgtcg 3120 cgctacgcgg tcaggccgat gccaagtccc gtacgggcat cgccgagctg accgttacgt 3180 agggcaccag cagcccgcgc gcccgggctg gatgacgacg aggatccgcg ggacttcacc 3240 cgccctcggc cgacagggac gtcctgacga gagcgagcac gtcgtcgtcg ctcagcccca 3300 gcgcgcgggc gtcggcgacc aggcgccggg cctgcacggt gagccgggcg cgctcggcgg 3360 tcgaccctcc ggtgacgacc gccccgcgcc cccggcgcag ttcgaggagg ccctcctcgc 3420 gcagccgttg gtagccacgg agcacggtgt gcatgttgac cccgagcgac gcggcgagat 3480 cgcgggcgga cggcaggcgc tcgccggagc gtacggtgcc gtcggcgatg gcaccacgga 3540 cgcatgcggc gatctggtcg cccaggggga ggggggacgc ggggtcgacg cggaagagca 3600 cggtcacccg cccgcggagg tgcggcgttc gcggtcggcg agggtgttga gcagcgcggc 3660 ggccgtggcg gcgtcgtcga cggtgacgac gaactcgctg ccggtggtca gacggacgct 3720 gatggcgtcg ccggaacgca gcacgacgcc gctcgccccg gaacggaccc ggtagcccca 3780 gccaccgaag tcccgcagag gcttgaccgg acggtgaccg gcttcggcga tccgctggag 3840 cggcacgttg atgcgcggcc aggggacggt cgagggcgtg acggtgagcc cgcgccggtc 3900 ggcggtcacc cggacacccg tcagcagggt catggcggct ccgatgagga acagcgacag 3960 cgcggacagc catccggcgg cgaccccgac gacgacgccg gaggcgaaga ccaggacacc 4020 ggtgaggggc agcacccggg agcccgccac ccttgaccag ccggcgatct cggagtcgcc 4080 gagcgcgaga cgcgaggcat cggcggacgg cccggaatcg ctgtcggctc cctggtcctt 4140 gccacaggcc gcccagccca ccgccgcgta gagcgcagca gccccgaaag cgagcgcggc 4200 ctgcgccccg ggcaaggtga cggaagaggc gtcgtggaca ccggtgttgg ccagcagcac 4260 ggcggtggcc agccatccgg tcagcaccgc gacggcgccg ccgatgacgg ccagcacgcg 4320 ctgtccgccg ttgcccggcc gcgtgaagta gacgagcgcg ccgaagagga caccgtcgcc 4380 cagcagcact ccccacgcga cgccgaggaa ggagccctgc cccgagaagc cgtcggcatt 4440 ccctcccggc ccgatgtgcg aggcgatccg cccgggcagc cggtcccgca ccgagaggaa 4500 cacccacagg acgacggcag cgcagaccag aggcggcacg acggagacgg caaggcgacg 4560 gacgaggacg gacgaggacc gtgacagcgg catgagagca aacctccact tgtttgcaca 4620 ctagtagaac aagtggaggt gaactcggcg aaggcggctg cctcttcctg acgcgttccg 4680 aacgccaccg gagccgccac gactgaccca gtgtcacccc gcgggaggcg gaacgcttca 4740 gtccgtgccg ggagcggcgg ccgcttcccg tggtgcgact ggtggtgtct gcgtggggcg 4800 gcgcatgagc ggcaggcgga gccgccggat gcccggcccc gagggaggtg ccagtctgcg 4860 cgtggacctg tggaagacga gcaggctgac ggcggcggct ccggcgaacc aggccagttg 4920 gacggcgagg tagccggaga cgggagcggt cgagaagccg gccgcggcac cggtctgcag 4980 cgagccgtag gaggggagga gcgtcacgag gccgccgttg gcggcgccgc cggtggtgac 5040 ggggttctgc aggcccgcgt cgagaccgct ggtcatcacg atggcgaaca tcccctcgac 5100 gtcccggcgc agcagggagc cgaagacgat gccgatggcg ccgtaggtca gggacgcgca 5160 gaacagggcg gcgacgaaga ggaccggccg gcggggcgac cagaaggcgt aggtgatggc 5220 ggtggcgtag acggcgacgg tggcggagat gagggtgagg gcggtgagct tggcgagaag 5280 gaggtggacg cgccggtagc ccgccatgga caggcgtcgg tcgaaggggc cgctggtgaa 5340 ggtcgcggcg aacatcatga agccgacgat cagggtgatg gagttcagcg ccccgacgat 5400 cgacgtgagt tcgttgcccc gcgggtggag gatctgcccg gtctcgtgca gcctgaaggg 5460 gatgggcggg tcttcgatga cgcagtaggc cagcgtgatc cacaccggga tgaacgcggc 5520 gatcaggccc atggcgagcc ggttgcgcag gtgctcgacc aaggcgaacc gggtggcggt 5580 gacgtagagg gttgtgtggt tcataccggt gccgtcgtcc ttgagtgcag cagtccgccg 5640 tcgaggtgcc ggagttcgtc gagccgttcg gcgtcgtagg ccaggtggga gacgaccagc 5700 acggaccgcc cgcgttcgcg cagaccggcg gccaggctcc agaaccgctg gtgggtgtcc 5760 cagtcgaagc cctggtaggg ctcgtccagg aggagcaggt cagggtcgtg catcagggcc 5820 agtgtgaggt tgagcttctg tttcgtgccg ccgctgagca cgctgacccg ctcgtccccg 5880 tagtcggaca gccggagcac gtccatgatt ctctcggcat ggctgagggt ggcgaggccg 5940 tacgccaccc ggaaatactc caggtgctgg cggacggtga gagcctggtt caggacgaga 6000 tgctgcgggc agtaaccgaa ccggccgccg tagtggacct gcccgcgctg cggccgcagc 6060 tcaccggcga ggatcttcag gagcgtcgac ttccccgcgc cgttctcgcc cacgacgccg 6120 gccagcgttc cggggcgcag ggacaagtcg atgccgcgca gcaccctgcg ggtgccgtag 6180 gtgtggtgga cgtccctgac gtccaggaga ttccggggca cggcttcctc tcctcaggcg 6240 acctggtcgc gccggacgac cgagagggcc agctcgtaca acaagtggct gtggcctgcc 6300 gacggcagca ggggctcgag cttgttccac gcgtcgctca ccaactggtc ggcttcctcc 6360 aggcaggctc tcaccgctcc gcaggcgttc aggtcccggc acacctcggc caccgccgtc 6420 gcgctgcccg agccgtcctt gacctgctgc cagagctggt tcagccgggc tctgcggcag 6480 ccggaccacg gcgtgggcca gtggcatggt gaccttgccg ctccgcaggt cctcggtggc 6540 ctgcttcgtc ggtgcccccg cccgtgtgac accgctcagg tcggcgacgt cgtcggcgat 6600 ctggtaggcg gtgcccaccg ctgaaccgaa agcccccagt gctctccgca gttccggctc 6660 ggcacccgtg acgacccctg ctgcctccat ggccgccgag accggggccc cggacttcaa 6720 ccggtgtgtc agacggacca gttccagcac agtgtgccgg tcgtcgccgg ccacggcctg 6780 gtccatctct tcccggtgac cttggagatc cagtgcctga ccggcgtgag ccgctcgcag 6840 cgcggccaga cccagtgccc gcaactcccc gcaccgcgag gcgtcgtcgg gaaaggtgag 6900 ctgaacggcc cgctcccaga ggaaataggc ggccgtaccc gcgttcaccg cagtcggcat 6960 gccgaacatg gtgtgcacgg ccggttgtcc gcggcgcagc ggtgaggcgt cctggacgtc 7020 gtcgacgatg agggatccgg tgtggagcag ctcactcgcc gcgatcagca ggccgcagga 7080 ttcgctgtca cctcccatca gaccgatggc ctcccaggcc agcaccggcc gccagcgctg 7140 tcctccggca tcggtcagat ggcggacggg agaggtcagg gcccggtgca gccgctgggc 7200 gacgaccggc ggcgtgccag gcccggtcgt cccggtgatg tggcccggtg gcagccacgt 7260 ggagcaagca tctgccgacg cgttcgggca caggcggtcg atgtgatggt tgatgcgttc 7320 ggccgtgcgg tcgatgcgct gccggatgaa gtccgcgttc gccgcgaaag tcggggagat 7380 gtccctggga cagaggaggg cgccggtcat ggagtggtca gctttcggtc aggggcgggc 7440 gatggacgag gctccctcat ggggtcgccg gcccgatgcc gcggagggac tgctcgggca 7500 cggctcgcgg agtgcggcga tcatggtcag gccgcgtggc atcgcggcga gttcgaagcc 7560 ggagcgcacc gtccggcccg gacgcgccgt gcgtacggtc cgcgtggcga ccagagtggc 7620 gagcgcgacg ggcaccatga cgtcggccac cgcggcacca gggcagtagc ggggcccgag 7680 cgcataggga agccaggcag caggggagac cgagggctga gcgtccggca tccagcgcgt 7740 cggatcgaag acatcaggcc gttcgaagtg ggcggggtca cggctcatcg ccccgaggtg 7800 gaggaacacc gtcgccttcg cgggcagccg gtggccgccc agccacgtct cgcggcgcgt 7860 gcaacgcaca aggaccggga ggccgtgaag ccggatgact tccttgacga aggcggctgt 7920 ccggggcaac cggtttattc cgacgccggc ctgcgagtgc cccaggccga gtgccgcgtc 7980 ggcctcctcc tgcaaggcct gttgatggtg ggggtggcgg cccagttcgt agcaggccca 8040 cgcgagtgtc gaggcggtgg cctccccgcc ggtgaacagc agcgtgcgca cgtcctgcac 8100 cgccgcttcc gagggctcgc gccacgcttg cttcagcagg gagaccacat cacacccgtc 8160 ctcagccggc cgatggcgcg cgaggacttc ccgggtggcc tcgtccagca ccgcgagggc 8220 acggcggagc gcgcgttgtc ttggcacggg tacccaaggc cacggggaca ggagaaaccg 8280 caaggccccg acctgcgaca gcgtggcatg cgccgcggcc agcgcggtca gcgttccggg 8340 cgagacctcg ctgcgcaaga cgcacctgac ggcgagatgg aaagtgagcc gggtcatctc 8400 gtggctcatg tcgaccggac ggtccgcggg aagggtggcg agcagacttc gggtctcggt 8460 ccgcacggac gccccgaggt ccgccggccg gggcacggcg aacgcgggcc tcatcaccgc 8520 ccgccggtca cggtggaccg ttccctcggt gctcagcagt ccctctctta cgatcacccg 8580 cacatggggt tccctgcccc agaacatgaa ggtgtcctcg tcgcgagcag cctgccggat 8640 cagcgaggag tcattgagca ggtacccgac gaacgggccc gccttcaccc tgaccacagg 8700 ccccgccttg ccgagccggg ccgcgatctc cctcagatcc atcaagaacc gcacggagcg 8760 ggcatgcccg agcaaccgac tgcagtcagg agccatcggc acaacggagt ccgccttcga 8820 atctccgttc atcaggcgtc ctcccgcccg catgtcaccc tctgtcctcc tgtgaacgac 8880 caggagtgag gagtgtcacg cagagcatca cctgctgtat cggcagcaat gccgacccgc 8940 accgacggct gggcggggcg accgggaacc gccttgcggc tacgccgtgc tcgcgtgcct 9000 gaagcgcacc gtcacattca ccggtaccca cgacagaggc gggttcgcgg ccacctgtat 9060 ggacgcccgc tgaatgggga cggagtgcag ggggtgctcg atggcggtgc cttcgcggct 9120 cagcaggccc tgcctcgcgg tttgagcgca acgggcggca gttcgggagc aggcagtcgg 9180 gccccctcgt agccgtgcac ctcgccgaag cgggagccgg tcgcccgagc cgtcgccgcc 9240 gccggcccga ttcgccatcc ggatggaaga ggacaccgcg cagggaccgc cgcccacttc 9300 accggaatcc tctccaccgg aaaatttatc cgcaaacctg tcacatcttc gacacatgaa 9360 gcgtcagggc ggtgacggca gttgaagccg ttgcccgacg acgccgaagg agaccgtggg 9420 acagctacgc acgtgcgggc cctggagcgg tcggccacgc cgacaggaat cggccttccg 9480 ccgctgatcc ggcctcccag cgttcgcgaa cacctcttgc cacacctccc gcgcggcccc 9540 cgcattcgag cggtcggctg acgacgccct gcgacgccgc gcacaccacc cacgcacctc 9600 gccacgccga aggctgcccg aaaacaagaa gaccgaggaa agcacacatg aagatctctc 9660 gaataggccg cgcgtcatcc atcgccgccc tggtgacaac cgcactcgct ttcacggcag 9720 ttggcaccgt cgctcccacg gccgtcgccg actcccgcgc ggccgccgct tccgggacgc 9780 agaatgacca cccgagctcg gggcagggca cctccacctc tgagctccgg cgcaagggcc 9840 tggtcccgtc gagtctcgtg gccaagccca tcacccgcag cgagaccctc agacgcgccg 9900 ccagctggtt cggcaagggt ctccactaca gcggggacaa cacctatcag ggctggcgca 9960 cggactgctc cggcttcgtc tccatggcct ggggactgcc cggcccgggt gagaccaccg 10020 attcgttcat tcccgggggc gtggcccacg aaatctccaa ggacgaactg aagcccggcg 10080 acgcgctcaa caacaaggcg ctcggcaacg acggtcacgt cgtcctgttc gagaagtggg 10140 ccgattcctc ccagtcctcc tactggggtt atgagttcag cagcagcggt ctgcaccacc 10200 gtgtgatccc gtacgcctac ttctccaggt ccgagcagta ccgcccgatc cgcttcaaca 10260 ccatcgtgga cgacgacacg gccgcagggc ccgccgagga caacgcccgg gtccagggtg 10320 acttcgacgg cgacggccgc gacgacgtgg cggtgctcta cgactacggc aggaaggacg 10380 accgcagtcg ctcggccctg tggacgttca acagcaacgg cagcggtttc aacagtccca 10440 agcaggtgtg ggacagcggg acgtcggaga gctggaactg ggcctccagc aagttgacgg 10500 tcggtgactt caacggcgac ggcaaggccg acatcggcgt cctctacaac atgggcgcga 10560 ccgaggacgg ccgcaaccgc accaagctgt tcgtgttcac cagcaccggc agcggattcg 10620 ccgccccggt caaggtctgg gacagcaacg acgaccccgt gaagagctgg aactggaacg 10680 ccagcaagct caccgtcggc gacttcaacg gcgacggcaa ggccgacatc ggggtgctgt 10740 acgactacgg caaggacgac gaccacaacc ggacagggct ctggacgttc accagcaccg 10800 gcagcgggtt caacagcccg aagcaggtgt gggacagcaa caacgacccc gtgaagagct 10860 ggaactggga agccagcaag cccgtctccg gggacttcaa cggcgacggc aaggccgaca 10920 tcggcgtcct ctacgactac ggcaagaccg actccggcag ccgcaccgga ctctggacgt 10980 tcaacggcaa tggcaacggg ttcaacagcc cgaagcaggt gtgggacagc aacaacgacc 11040 ccgtgaagag ctggaactgg gaagccggca agcccgtttc cggcgacttc aacggcgacg 11100 gcaagagcga catcggcgtc ctctacgaca tgggtcgcac cgaggacggc cgcaaccgca 11160 ccaagctgtt caccttcacc ggcacggcga ccggtttcaa cagcccggtc aaggtgtggg 11220 acagcaacga cgaccccgtg aagagctgga acgcgtccgc gagcaagccc gtcgcaggtg 11280 acttcaacgg cgacggcaag gcggacatcg gcgtcctcta cgactacggc aagaccgact 11340 ccggcaaccg cagcggactg tggaccttca ccagcaacgg cagcggcagc gacagcccca 11400 agcttggctg ggacagcagc gcggaccccg tcaagagctg gaactggagc gcgagcaagc 11460 tcggctgacc ggcttcgccc ctcctcacct caccgttcgg gagagtcacc gcacatgcga 11520 accatacgaa tacgaagaac gaacggcgtg gccttcgccg ccgctgccgc cctgatggcc 11580 ctcgtcgcct ccggcaccgc cacggtccag gccgcgccct cgcacgccgg accctccggc 11640 accactccga tcacctaccg tggcctcacc ctcgacatac cctccgggtg gccggtcgtg 11700 gacctggaga aagacccgca cacgtgtgtg cggttcgacc gccacacggt gtacttgggc 11760 caccccggca ccgaacagtc ctgcccctcc catctggtcg cggacaagac ggacgccctg 11820 atattggagc cgatcaccgg agcgggcggc caggacgcct cccacgcgct gcgcatccct 11880 gccggggccc cgatgccgca cgagctgccg gtgacgtacg accacgagac gaaggtcgcc 11940 gtcgaaggcg ccggagtcat ggtcacgtcc tcctacggca cgtccagtac aacggtcgcc 12000 gccgtcctcg gctcggcccg cacggacgcg acagccaagc cgacccccct gcccggcaag 12060 gcgggcaggg gcctcgcggc tccaccggtt gccgccgtcg cggccgacaa gggatacaca 12120 gggctgggct tcgagtcctg caccgcccct tcgtccgccg cgatgaaggc atggaaggcc 12180 tcgtcgccct acggggccgt cggcatctac atcggcggtc gcaagcgggg ctgtgcgcaa 12240 ccgcagctca ccggcgactg ggtgcgtcag cagaccgccg acggctggca cctgctgcct 12300 ctcttcgtgg acctccaggc cggcgacatc tctccggcca ccgcggacgc gcagggccgc 12360 gagtccgcgg acgccgccgt ggccaaggcg gcggacctgg gcctgggccc cgggacggtc 12420 atctacagcg acatggagca ctacgacagc cgctcgtacc gggcccgggt catcgactac 12480 gtgtcggggt ggaccagccg cctccacgaa catggctacc gctccggtgt gtacgcgggt 12540 gaaacgagcg gcatcccgga cctcgcctcg gtggccgacg acaccaacta cgcatcaccc 12600 gacgtgctgt ggtcggcgaa ctggaacctc aaggccgatg tgtcggacgc gtcgatggga 12660 cttccgggcc ccggctactg gcccaatggg cggcgcatcc accagtaccg cggccaggtg 12720 aacgacacct acggcggtgt caccctcgcc atcgaccgcg actacgtcga tgtcgccgcg 12780 gactcggccc tgcccgcacc cggcggagag gacggttcct cgcgcgtcaa gggcgacttc 12840 gacggcgacg gccgcgacga cgtggccgtg ctgtacgact acggcaagga gggcggcgtc 12900 agccggtccg cgctgtggac gttcgcgggg accggcagcg gcttcggcgc cccgaagaag 12960 gtgtgggaca gcggatcgga cagctggagt tggtcggccg ccaagctgac ggccggcgat 13020 ttcaacggag acggcaaggc cgacatcgcg gtcctgtacg acatgggtcg cactgaggac 13080 ggccgcaacc gcaccaagtt gtacgagttc accagcaccg gcagcggatt caacagcccg 13140 gtcaaggtct gggacagcaa cgacgacccc gtcaagagct ggaactgggc ctccagcaag 13200 ctgaccgtcg gcgacttcga cggcgacggc aaggccgaca tcgcggttct gtacgactac 13260 ggcagggacg gcgaccgcag ccgtacgggc ctgtggacct tcaccagcac cggtgccgcc 13320 ttcaccggcc ccaagctggt gtgggacagc aacaacgacc cggtcaagag ctggaactgg 13380 aacgccagca agcccaccgt cggcgacttc aacggcgacg gcaaggccga catcggcgtc 13440 ctctacgaca tgggtcgcac cgaggacggc cgcaaccgca ccaagctgtt caccttcacc 13500 ggcacggcga ccggtttcaa cagcccggtc aaggtgtggg acagcaacga cgaccccgtg 13560 aagagctgga actgggacgc cgtcaaggta gtgggaggcg acttcaacgg cgacggcaag 13620 agcgacatcg gggtgttgta cgactacggc aaggacggcg accgcagccg caccggactg 13680 tggaccttca ccagcaacgg cagcgggttc aacagcccga agcaggtgtg ggacagcagc 13740 aacgacccgg tgaagagctg gaactgggcc gcgagcaagc cggtcgcagg ggacttcaac 13800 ggcgacggaa aaacggatat cggcgtgctc tacgactacg gcaggaccga ttccggcaat 13860 cgcaccggac tgtggacctt caccagcgac ggcaccggat tcggtacacc cctcctgggc 13920 tgggacagcg tgacggatgc cgtgaagagc tggaactggc gtgccagcaa ggtgagttga 13980 cacccctcct gtgagacatg gggcactcct cgacgcccgt ccggcccggc tgcggcccgg 14040 ccggacgggc ccgtcattca atggaaggaa gaagtggatc ccttgacgcg caagacccgc 14100 accccccgca agaagggcag acgcgcgagc gcggcggcga tgtcggcctc cggcatgctg 14160 ctcgccttgg tggccaccgc cgcccccgtc cccgcccagg cggcatcact cgccacctgg 14220 gaaaagatgg cccagtgcga gagcagcggg gactggggat acaaccagcc accgtactac 14280 ggcggcctgc aattcctgga gagtacgtgg gtggcgtacc acggaacgga ctatgcgcca 14340 tacccctatc aggccaccaa ggaacagcag atccgggtcg cgcagcggct cctcgacaat 14400 gagggcgcgg ctccctggcc gtactgcgga aagaaggtgg ggctggctga cgacgacgca 14460 cgccccttcc ccgacgcgcc ggacgacgac gcctccgccc ggatcaacgg tgacttcgac 14520 ggcgacggat gcgacgacgt ggccgtgctc tatgactacg gcaaggaggg cggcgtcagc 14580 cggtccgggc tgtggacgtt ctccgggagc ggtaccggcc tcggcagccc gaagaaggtg 14640 tgggacagcg gatcggccag ctggagttgg tcggccgcca aactggccgt cggcgatttc 14700 aacggcgacg gcaaggccga catcgcggtc ctgtacgaca tgggccgcac tgaggacggc 14760 cgcaaccgca ccaagttgta cgagttcacc agcaccggca gcggattcaa cagcccggtc 14820 aaggtctggg acagcaacga cgaccccgtc aagagctgga actggaacgc cggcaagctc 14880 accgtcggcg acttcaacgg tgacggcaag accgacatcg gcgtcctcta cgactccggc 14940 aagaccgact ccggcaaccg caccggactg tggaccttca ccagcaacgg cactggattc 15000 aacagcccga aacaggtgtg ggacagcaag agcgacccgg tgaaaagctg gaactgggcc 15060 gcgagcaagc cggtcgcggg cgatttcaac ggtgacggca agaccgatat cggggtgctt 15120 tacgactacg gcaaagatgg cgaccgcagc cgcaccggac tgtggacctt caccagcacg 15180 ggcagcggat tcaacagccc caagcagacc tgggacagcg ggtcggaaag ctggagatgg 15240 tcggcggcca aggtggtcgg cggcgacttc aacggtgacg gcaaggccga catcggggtg 15300 ctgtacgacc tcggcaggaa cggcgaccgc aaccgcaccg aactgttcac gttcgcgggc 15360 aacggcaccg gcctcaacac accggccaag gtgtgggaca gccaggacga cagcgcggtg 15420 aagagctgga actgggccgc gagcaagccg gtcgcaggtg acttcaacgg cgacggaaag 15480 acggatatcg gcgtcctcta cgactacggc cagaccgact ccggcaaccg caccgggctg 15540 tggaccttca ccagcgacgg cagtggattc gccggcccca agctcacctg ggacagccgg 15600 accgaccccg tcaagagctg gaactggaac atgagcaaga ccggctgagc cattcatgcc 15660 gtacagaaga gaagaggaag gatgaaatac cgaccgggaa cactgctcac ttccataaca 15720 gtcttgtgtg ccctgctcgt tccggtgcgt tcggcggctc aggcggccag gcccgagcag 15780 ggacgttccg tggtggccgc ggccgccgta ctggagcaaa gtccgccgac gctgctcgcc 15840 gagccggaaa tgcgcgtcgt ctcctggaac atctgcggtg aggcgggcgg ggtgcgcggg 15900 gagggcggct actgccccta ccgcaacgat ccccaggcga aagtcgacca gatcgcgcag 15960 gtggtcgcgg agcgcagtgc caatgtcgtc atgctccagg aagtgtgcgg cgaggcgccc 16020 ggcagccata tggagcggct gcgcgcggcc ctgggcagcg gatggtcgat cgcgcacgcc 16080 ccgggggccc gcccggacga cggaaccacg aactgccggg gcgggctcag cggcatattg 16140 ggcgtgggga tcgcggtgaa ggggcgcgtc accgacacca ccgcgacgaa caccgtgccc 16200 gggggcggcg gtgacaagca gaccctgccc atcctctgtg tacgtgtcga gggctggtcg 16260 tccaggatct gcaccaccca catcctgtcc gaccctgccg atccgcgcag gccggggcag 16320 atccagaacg tcaagaacga gatctggccg gaccgctatc agctggtgct cggcggcgac 16380 ttcaacatgt tccccgactc cgccgggctc aagccgatct cggacgaatt cgacgagtgc 16440 gaccgccgct cctacggcgc cggtgacatg gtcaacgagg tcacccatca ctcctgggag 16500 aaaaagggcg gacacatatg gcgcaagcgt gaccacatct tcgcctcgtg gggagagtcc 16560 gggagccagt tcacatcctg cgaggtcgac cggacccgga tggacaccac cgagaacgcg 16620 cccgaaagcg gtccgcccaa cgggtattcg gaccatgcgc cgatcatcgg ctacctcaag 16680 ccgccgcggc acctgagcac gtccggggac ttcgacggcg acggcaaggc cgacctcgcg 16740 gtcctctacg ggcaggggaa gaccccggac ggccacaacc ggtccagcct gtggatctca 16800 ggcggttccg gtaccggagc ggagaccgga ttcgccgcgc cgcgcgaggt ctgggacagc 16860 ggtgccgaca gctggaactg gtccgcgagc gcgctgacct ccggggactt cgacggcgac 16920 ggcaagaccg acatcggcgt cctctacaac tacggcaggg acggcgaccg caaccgcacc 16980 gcgctgtgga ccttcaaggg gacatcgaac ggcttcgagg cgccccgcaa ggtgtgggac 17040 agccacgacg acacggccgt tcccagctgg aactggtcca cgagcaagct cgtcgcgggc 17100 gatttcaacg gcgacggcaa agcggacatc ggcgtcctgt acgactacgg caggaccgcc 17160 tccggcaacc gcaccggact gtggaccttc accagcaccg gcaccggatt cggcaagccc 17220 cacctggcgt gggacagctc caccgacccg gtgaagagct ggaactgggc cgcgagcaag 17280 ccggtcgcag gtgacttcaa cggcgatggc aagaccgaca tcggcgtcct ctacgactac 17340 ggcaaccaca ccgccctatg gaccttcacc agcaacggca ccggattcgc cggccccaag 17400 caggcctggg acagcggacc ggagaactgg aactggtccg ccgccaagcc ggtcgccggg 17460 gacttcgacg gcgacggcag gaccgacatc gcggtcctgt acgactacgg caggaccgcc 17520 tccggcaacc gcaccggact gtggaccttc accggcaccg gcaccggatt cggcaagccc 17580 cacctggcgt gggacagctc caccgacccg gtgaagagct ggaactgggc cgcgagcgag 17640 ccggtcgctg gtgacttcaa cggggacggc agggccgacc tcgcggtgat gtacgactac 17700 gggaacgcga ccaacggccg caaccgcacc gcgctgtggt ccttcaccag ccgcggcacg 17760 gacttcgccg ccccgcgggc gaactgggac agcagcaacg ccgctgacca gctgaaatcg 17820 ggcgagctga gggcggctcc gctcagcggg tcctagttct ccatgatcgg tccgtcgccc 17880 tccagaccgg ccgctctccc ggtcagcgtc gcggccagtg cgtcagcgtc gcgaccgagt 17940 ccgtaacagc gcatcccggc gatcgcgaag tacgcctggt cgagccagac gcgggccgcg 18000 ccagtgctgc cgcgcggcga agtacggcga gctc 18034 76 53500 DNA Streptomyces lavendulae 76 gtccgggccg gcggcaccgg ccgagccact gctcgtacgg cagtcctgag ggctcacggc 60 cacccgaaga acgcgcggta gcacggaacc tgctgctcca gcatatggat gccgtggtgc 120 acacggcgcc cggcggtggc ggccgcgctc agcagcgccg tctcgtgcgg cttcatgacg 180 acgtcgacca ccacggcatc cggtcgcacc ctcgcggggt cgaagggcag cgggtcctcg 240 gaacgcatgc ccagaggcgt cgcgttgacg gcgaaatcgg ccgcctccag atcgccgggc 300 cccagcgccc ggatcccgtc cggccggcgg gacccgagcc gcagcagcag cgcgtcgagc 360 tgggcgcggt cggtgtcgtg cacggacacc cgcgcggcgt cggccatcag cagcgccgtg 420 gcgatcgcgc tgcccgcccc tccggcgccg accagtgcca catgcctgtc gcgcaccgtg 480 tgcccggccg cctgaagacc ctggacgaac ccgagcccgt cgaagttctc ggcgtaccag 540 cggccgtcgg gttcgcgccg catcgcgttg gccgtcccga tgagggcggc cgccggcccg 600 agcccgtccg cgagcccgca cagggccgcc ttgtgcggca cggtgaccag cagaccgtcc 660 agattgccga tccgcttgag cccctcgacc acctcggcga gatcccgcgc ccggacgtgc 720 accggcacca ccacggcgtc cagaccgctt tcgctcagca gggggttgag cagaccgggc 780 gccttgacct gggcgacggg atcacccagc accgcgtaca gccgcgtggc gcccgagaca 840 ccggccgccg gcccgaggaa ttccatcagc cgatcctctc tgtacccccg acggatgttg 900 ccctacggtg ctggagatgc tccacagctt tgccgtgacc gccggtcggc acaaccctgc 960 gtgcccctga cgcgccaggc cctccaggta gttgctcccg gcggatcccg acagctcccg 1020 accggtcccg acggagggaa gaagccatca gatacctggg aatcgacgtc ggaggcacga 1080 aggtcgccct gcgggtgacg ggggacaccg acggtgcggg cggcggcgac gtgacgttcc 1140 gctggcccgc cgccggcgac gtcaccgcgg atctggacct gctcgccgcg cgggtccgcg 1200 gtcttctggg acaccgcgag gaccccctcg ccggggtcgg cgtggccatg cccgcgatct 1260 gcgacgcggc cgggacggtc cgcacgtggc cgggacggcc gagctgggcg ggcctgaacc 1320 tgacggccgc cttcgggcag ttgctgcccg gcaccccggt cgcctgcgcc gacgacggtg 1380 acctggccgc gctggcggag tcccgcgccg ccggctgccg gcatctgctg tacgtggggg 1440 tcggcacggg catcggcggc ggcatcgtcc atgagggccg cgcctggccg ggccccggac 1500 gcggctcgtg cgaggtcggc catgtcgtcg tcgaccgctc gggcccacgc tgcgactgcg 1560 ggcgcgccgg ctgcgtccag gcggtcgcgt cgggaccggc gaccctccgg cgggccgccg 1620 aacggcgcgg ccgggagacc ggcttcgacg aactggcctc cggggcgcgc ttgcacgccc 1680 cgtgggcgga agcggccgtc gacgagagcg ccgcggccct ggccaccgcc gtgaccggca 1740 tctgcgagct ggcccacccc gaactcgtcc tcgtcggcgg cgggttcgcg gcgggcgtgc 1800 cgggatacgt ggcctcggtg gcggcgcacg tcgagcggct gacccgcccg ggaacggatc 1860 ccgtgcgggt gcgcccggcg gtgctcggcg ggcggtcctc cctgcacggc gcactgctgc 1920 tcgcgcggga ggcacacggg cggggaaacc ggccgccgga gagtgaccgt gtttcttccg 1980 atgtttcttc cgatgtttct ttcgggggag tgacagacag ggccgttggc cggtccgact 2040 gagcacaatc acaggtgatt tcgcccaggt tcaccacgcc tcgtgtgctc ggggtcggca 2100 gaaggagtca gagtcatgct cgacaggcgg agcgtcattc gcgtcggcgc cggggtggcg 2160 gcggccgccg ccgtggccgg tacggccgcc accggtgcgg cggccgtggg gctgccgggt 2220 gtacggggac gcgcggcgtc gcgcggggtc gactgggcct ccttacgccg tcatctgtcg 2280 ggcgagctcg tcctgccggc ggacaccgga tacgagcggg ccaggaagct ctacagcggc 2340 cagttcgacg gcatccgccc gcaggccgtc gcctactgcc ggaccgagga ggacgtgcgg 2400 acgaccctcg cgttcgccca ggaccacgcg ctgcccctca ccccgcgcag tggcgggcac 2460 agcttcggcg gctactccac gaccgacgga atcgtcctgg acgtctccgg cttccacgcg 2520 gtgagcctca cccggaacac cgtcgtcatg ggcgcgggca cccagcaggt ggacgccctc 2580 accgccctgt cgccgcgcgg tgtcgccgtg gcgagcggca actgcgcggg cgtctgtccc 2640 ggcggcttcg tccagggcgg cggactgggc tggcagagcc gcaagttcgg catggcgtgc 2700 gaccggctcg tctccgcccg ggtcgtgctc gccgacggcc gcgccgtgac cgcctccgcc 2760 accgaacacc ccgacctttt ctgggcgatg cgcggcggag gcggcggcaa cttcggcgtc 2820 gtcaccggct tcgagctgcg ccccaccgac gtcccctccg tcgtcagcta caacctcacc 2880 tggccgtggg agtcggcgcg gcgcgtcatc gaggcgtggc agcactggat catcgacggc 2940 ccccgcgacc tcggtgccgc gatggccgtg cagtggcccg acgccgggac cggcacgccg 3000 gtcgtggtcg tcaccggcgc ctggctgggc gcggccgacg cgctcacccc cgtgctggac 3060 tccctggtgg cctccgtggg cagcgcgccc gccacccgct cggccaaggc gctctcccag 3120 cacgacgcga tgatggcgca gtacggctgc gccgacctca cgcccgagca gtgccacacg 3180 gtcggctact cgcccgaggc cgcgctgccc cggcagaact tctccatgga ccgcaaccgg 3240 ctcttctccc gggccatcgg gcaaggaggc gtcgagcgga tcctggaggc gttcgccgcc 3300 gacccgcgcg ccggacagtt ccgcttcctg agcttcttcg ccctcggcgg cgccgccaac 3360 cgccccgacc gcaccaccac cgcctacgtt caccgcgaca ccgagttcta cctcggtttc 3420 tcgatcgggc tgaacgaccc ggagtacacg gcggaggacg agaggctcgg ccgcgcctgg 3480 gccgcgcgag gactgcgcac gctcgatccc cactccaacg gcgagagcta ccagaacttc 3540 atcgacccgg agctcgacga ctggaagtcg gcctactacg ccgagaacta cgtgcgcctg 3600 gccgccgtca aggcggccta cgacccgcac cggctcttct ccttcgcgca ggccgtctga 3660 cctctcccga aagacccctg ccggcctgct cccctccgcg gctcctgtgg gcactggtgc 3720 gcccgcgcac ttctgtgtga ttgagtgaag tccgggcgtg cagagctcag ttgccgtgga 3780 gggggcgcca gttgcgagca tcagcggtgg agagggtgga gctgatccgc tggccggtgg 3840 agtccgagcg gcgggagcgc tgccgcgacc ggggcgtcat gcggatcctg gtgctggagg 3900 cgggggccga ggcacccttg tgcgtggacc ccaaggagga ctgggtccgc gctcccgtca 3960 gcaccgacga cctgcgggcc cgcgtcgagg ccctgcgcct tcggggagcc gccgccgagt 4020 cccggcccga ggtcgacccg aacggagtgc tgcgtttccg gtggcgctcc gccctgctct 4080 cgcccaccga ggcccggctc gtcgcccggc tcgccgagtc ctatgccgag gtcgtcgccc 4140 gcgacgacct gctccgcccg cccccgggcc gtaccgtgcc gagccgtaac gcgctcgacc 4200 tccacatcat gcggatccga cggcgcctcg ccgcgctggg cctgagggtg cgcaccgtcc 4260 gggggcgtgg ctacgtcctg gagagcgcgg aaggagtctg accgacgggc gtggccgcgc 4320 accgcaccga ccgcccctac gagcgaggag cccgaagtgc agcagcctca tcacagccgc 4380 gtcgacgtgg aactgggcga gaggtcctac cccgtccacg tcggaccggg ggtccgccac 4440 ctcctgcccg gcatcgtcgc ctccctcggc gcgcaccgcg ccgccgtcgt gaccgcacgg 4500 ccccccgacc tggtgcccga tcccggcgtg cccgcgctga tcgtgcgggc acgtgacggc 4560 gagcggcaca agacgctcgc caccgtcgag gacctgtgcc gcaagttcac caccttcggc 4620 atcacgcgcc acgacgtcgt cgtctcctgc ggaggaggct cgacgaccga caccgtcggc 4680 ctggcggcgg cgctgcacca ccgtggggtg ccggtggtgc acctgccgac caccctcctg 4740 gcccaggtgg acgcgagcgt cggcggcaag acggcggtca acctgcccga gggcaagaac 4800 ctcgtcggcg cctactggca gcccaaggcc gtgctgtgcg acaccacgta tctccagacg 4860 ctgcccgccg aggagtgggt caacggctac ggcgagatag cgcgctgcca cttcatcggt 4920 gccggcgacc tccgcggcct cgccgtccac gaccaggtca ccgcgagcct gcggctgaag 4980 gcgtccgtcg tcgcggccga cgagcgggac accggcctgc ggcacatcct caactacggc 5040 catacgctgg gccacgcact ggagaccgcc accggcttcg ggctgcggca cggactcggc 5100 gtggcgatcg ggacggtctt cgcgggccgg ctcgcggagg cgctgggccg catcggcgcc 5160 gaccgcgcgc gggagcacac cgaggtcgtc cgccactacg gacttcccga cagcctcccg 5220 ggaaacaccg acatcaccga gctcgtcgcg ctgatgaggc acgacaagaa ggccacgtcg 5280 ggactgacct tcgtgctcga cgggccttcc ggcgtggagc tggtgtccgg gatcccggag 5340 gacgtcgtcc tgcgtacgct cgcggcgatg ccgcgaggaa cggcctgacc gagtgttccg 5400 tcttccgagg ggaagtgacc gtttcgtgtc ggcagagctg tcagaaccgc tgaagaaggc 5460 cctggactcc ctggtgttcg gcgtcgtggc gacgaccgac cccgacggcc gcccgcacca 5520 gtcggtggtg tgggtccggc gcgagggctc cgacgtgctg ttctcgatca cgcgcggcag 5580 ccgcaaggag aggaacatcc tgcgcgaccc gcgtgtgagc gtgctgatca gcccggcgga 5640 ctcgccgtac acctacgccg cgatccgggg caccgcgcac ttcgaggacg tgccggaccc 5700 gggcgcgtac ctcgacacgt tctccataaa gtaccacggc gtgccctacc gggagtcgtt 5760 ccccgagccg ccggaggtga gcaccattct cgccgtccgg ctcgttccga cgtcggtcta 5820 cgagcagtgg tgagggcgta ggcgtcccga agccccggca gcgtcccgaa tgccgctgcc 5880 ggggcttccc gtgggagccc tacgccggtt tccgcgcggt gaccaccgag tagccgacct 5940 cctccaccga gcccatgcgg tcgatgccgt cgaccatgcg gtggaacgcc tcgtcgtcca 6000 tgtgggagcc gagctcgtcc ctggccgcac gcatcttcgc cgccaccgcc tcgtaggagg 6060 gccgcacctc gtccccgatg tcgaggaact ccaccacctc cagccccacc gaccgcatgc 6120 agtcctcgta cgcctcgcgg gtgaggacgg ggccctgctg gaagttgtcg ttggcggtgt 6180 cgacgatcgt cctggacgcg tcgctcaggg gccggcgcag cacgaagtcc gtcaccgtca 6240 cccggccgcc cgggcgcagc acccgggcga tctcggtgaa gacgtcggcc cgttccaggg 6300 cgtggcagat gctctcgatc gcgtaacagg cgtcgaacga gccgtcggga aaggggagcg 6360 cgagcatgtc accgatgcgg aactcggtcg cctcgtcgcc ctccttctcg gcgagctgcc 6420 gcgacagacc cacctggtag gggttgatgg agaccccggt ggcccgcacc ccgtgccggg 6480 cggcgatgcg caaggtggcc ttgccgttgc ccgaccccac gtcgagcacc cgctccccgg 6540 gggcgaggcg caggcgctcc gagacgtagt cggtcagccg gtcgcctgcc tcttccaccg 6600 tcgtggggac gtcgggtccc gcccagtagc caccgtgcat gtagccgcct tccgcgtgca 6660 ccatcaagtc ggtgacgcgg ttgtagagtt cgaccatccg gtcggaggcg gacgcggttt 6720 ccgtcatgcc gctcactttc ccgggcgctg ggcgaccagc agcagatagc cgaactcctt 6780 gacgccgacg aggtcgccgg ggtcgaactg gttcaccatc tcctcgccga actgcgtctc 6840 cagcctctgc ttcgaggagt tgatgcgctc cgagagcagc ctgaaggtct tctccagggt 6900 ctggtcgctg atgtcgagga actcctccag ccacaggccc gccccccgca gcaggggagg 6960 gtacgcctcg gcgctgacca tggtcatcat gaagtcgtgg aggtagcgct ggacggcggc 7020 ccgcccctcg ggggcgaggg gggcccgctc gaagaagtcg gtgagcacca gacggccccc 7080 gggccgcagc acccggccga cctgggcgag cacctgggcg cggtcgggca tgtggatgat 7140 cgattcgagg gcgatgacgg cgtcgaagct ctcgtcctcg aaggggaggt ccatcgcgtc 7200 ggcccgctgg aagcgcgccc ggtcggcgag cccggcctcc tcggccagcg cgttggcccg 7260 gacgacctgc tcatggctca ccgagatgcc cgtgacatgc gctccgctga gccgggcgat 7320 gcgtacgccc ggggtcccca cgccgcagcc gaggtccagg acgcgggagc cggcgccgat 7380 gcgcagccgc tcggccatca tgtcggtgag ccggtcggtg gcctcggcca gcggcacctg 7440 gctgtcgggg gagtcccagt agccgaagtg caggttctcg ccgagggagg cggctcccag 7500 cgcggtgaac cggtcgtaga gcgcgcccac ttcctcggag gcgggtgagg gcatggggag 7560 ttcggacagc tcggagtgcg gcatggacga tccctctcgt gaaaggtcgg gggtgggtcg 7620 ggcagtcggt gtcaggagag acggaggtcc tggtagacgg cgtcggcgag gcggacgatg 7680 cgctcgcggt gccgttcgtg ctccaccttc ccgttgaggt ggagggcgac gacgagagcg 7740 gcgtccgggt cggcgaagac cacggcggtc cacagcccgt cgtgcccgaa ggaccggggg 7800 gaggcgtacg agccgaagct ggtgaaccgc ggatccagct gacggcattc gaggcggaac 7860 cccatgcccc agtcggcgtt gccgtagcgg tcctggaggc cggtgcggtg ccgggccgtg 7920 agggcggcga cggtgggcgg cgccaggacg cgcccgccgg gagcgtcccc gccgcgcagc 7980 agcatctcga agagcctgcc catgtcccgc agcgggccac gggtgttgac ccccgggatg 8040 cagcgtgtgg tggccgcctc cgtcgaccac cagtgggtgg gcagcgggcc gccctcgggg 8100 ttgctcacat ggatcagcgg cagctcgccc ccgagcgcgg cgaactcctc gcgatccagg 8160 tggacacggg tgccggacat gccgcacggc ccgaggatct cctcctggac gtacgcgcgg 8220 tactccctgc cgtcgacgac cggaaggatg cgcgccagga cgaaccaggc ccaccactgg 8280 ctgtagttga tgccgggcgt gccccccgga cgcggtgcca ccggcacctc gaaggcacgg 8340 cgcacacgct cctcgtccgg gccggccacg atgccgtgca gcgggtcgtc gccggtgggc 8400 agcgggcccg tatgcgtcag cagttccatc gaggtgatgg actccttgcc ccggttgccg 8460 aactccggca gatagtgcgc gacgggcaga tacgggtcgt acgctcccgc ctcccacagc 8520 cggcccaggg cgaccgacag cagtggcttg gcgcagcagt accacagggg cagcgaccgg 8580 tgggtcatcg ccaccccggg gcgggccagc cccaacccgg cgtccgccag agggactccg 8640 tcgcgggaga cgtagatctg cgcccccggg gtcgaggtgc cgacctcgcg ctccagctcc 8700 cgcatggtgc cgggaagcgc cgcacgggcg cgccggacgg cctcggccgc gtcctcggtg 8760 ccgggcggcg gggccgcttc ccgcgccgtg gtaccgggcg tgcccctgag cgcggccaca 8820 tggcccgagg cgtcccgctc cagtgcggcg tcgatgaaga acagcaggct gaaattgccc 8880 tcggcccgta cgtcgccccg ctcgaatacg ccggtccgat cccgcgcggg gccgagcagc 8940 agggcgcggg cggcctccag gggaagccgg atccgcagtc ccggactctc accggccgcg 9000 gccgcgaggc cgagccgccc ggcgacgagg tccacctgga cgtcgacggc cgggggttcg 9060 ccgggcggtg ggtcggtgag ctccaggcgg agcgcgaccg tgccgcgttc gcccacgggc 9120 gggcgttcga gcctcgcggc cccggcgagc caggccgtgg acaggaacgg cgcggtggac 9180 agggtgttca gcacgggctc actcctccgc ttccttggcg gcccccggcg ccacctgggg 9240 gacgacctcc tggaacagcc gctccaggct ggtggtggcc gggtagtcgc ggaaccacag 9300 cacgaactcc gccacgcccg cggcgaccag gcgccgggcg cgctcggtga gctgttccgg 9360 cgtcccgtac aggctgcgcc gcgccagcag gtcgggatgg tggctccaga acagcacctc 9420 gtgcggggtg gagagccagc ggtcgcgttc caggacggag tcgaagatcc ggcactccgc 9480 ggcccaggcg tgccggacgc cgtccggatc gagcccgagc tccgtgcgac ggcggcggaa 9540 cgcggtgacg gccgcggcga cctgagcggg ctcaccggtc cactggacat gagcgcactc 9600 ccgcacggtg gcgtcggcgg gccgcagcgc gccgctcccg gcgtccccgg ccggggtgcg 9660 cagcgcgagg gggaggggct gctgccgtgg ggcgggcacg cagtgcgccg aagtgaggcg 9720 gatgtgttcc ccggtgaagg tgacgggctg tccgccccac agcgcgcgca gggcctcgac 9780 cgtctcgccg agagcccggt ggccggcggc ctcctcctcg tccgcctcca ggcccgtggg 9840 cacctcgcgc cccgtcgagt ggtgctccgg caggtactcg cgggccggga agccgagggt 9900 gagccggccg tcgcagacga cgtccagggt cgcggcccgc ttggcgatca gcgcggcgtt 9960 gcggaacggc ggggccgagg agagcagtcc cagatccaga ccgggcaccg cgcccgcgag 10020 ggccgccagc gccgtccagc cctcccagac cggctcgggc tcgcgccggg gcagggtgtc 10080 ggtgcggtcc agcagccaga gggcgccacg gccgtgccgg tggacggtgc gggcggtctc 10140 caggaggagc cgccagccct cgcggccgcc cgtaccggcc agttcgaggt tggtgccctg 10200 cggggcgacg acgctccagc gcggggtcgc gggcgtcatg cgtggccgcc gctgcggacc 10260 agcgcgagca ggctctgcgg cttgtccagc aggaagtcgg gccgctcggc gcgcagttgg 10320 ccggccgcgc cctcgcccca ggtcgcgccc acggtggcgg tcccggcggc gcggccgctg 10380 cggatgtcga tcaccgcgtc gccgaccatg accgcgtcct cgggcgccgc gtccagccgg 10440 cgcagtgcct cccgcacgat gtcggggtgc ggcttgggcc tcggcacctc gtcgctgccc 10500 accacctcgt ccagcagggg cagcagcccg accgcctcca gcacggcgcg cgcccgggag 10560 ccggacttgc cggtggcgat cgcggtgccg acgccgtccg cgcgcagctc cgccagcaac 10620 tgcggcacgt ccgggtacac ctcgacgcgg tccatcagcc ggtggctctc ccggacgaac 10680 ggttcctcca tctcgccggg caggcccatc agccgcatga tgtccgggaa gtagcggccc 10740 tggtgcgtgc ggtactcctc gaagggcggc tcgcccggcc ccaccacttc gcggtaggcc 10800 acggcgaacg cctcgcgcat caccgcgaaa ctgtcgatga ggacgccgtc gaggtcgaag 10860 accacggtgg tgaaccggca cgggagagcg gtggcgggcg cgtccgccgc ggcccgcgag 10920 gcgggcgggg ggttcagggg catggcagga gctccgtggg tagggacgtc ggggcccaga 10980 ccccggcgtg gctgacgact ttcgtgaagc ggcccgcgag ccggtggtag acggcccccg 11040 gcgagctgtg cggcgggtgg gccgccgcgt ccagcccgcc ccaggcggcc atggccctca 11100 gcagcagcgg atcggccggc acccagcggc cgcccaccag gaactcggcc cagcagtgcg 11160 gtgtggagta cggcttggcc accagcagcc cgaaggagaa ccgcacgtcg agcccgcgcc 11220 gccggccctc cgccaccagc caggccgcgg cgccgccgca gtcggccatg tgcgtgctcc 11280 acaggaaccc cgggtcccag cggatcgctt cgggcagcag gaagaagccg accggctcca 11340 gggtgcgcag caggtcgagg acggccgggg ggaagtccgg ccagtggccg cgcgaccgtg 11400 tgtacagccg cgccagcgtg gtctcccgcg gcgaagccgc ctccaccggg cggcgggcgc 11460 cgggcagcag cactccgtag cggcacgggc ccgcgtggcc gggcaccggg caggacgccg 11520 tgacgtcgac gcgccagcgc gggctctccg cggccgaggc ggtccgcagg gaaccggccc 11580 acgagcggat ggcccggcgc tgcaccgagg acaggccaag gtgcagggcc gcgttgccca 11640 ggtcgtagcc gtcgaagagg cgtcccgcgc cgcttcccac gaagggcacg cccgcctcgc 11700 acagcgcgga cagcagctcc ggtccgatcc ggtgcagccg gcgcgcgctc tgctcgtcga 11760 cggtgaaacg gcggtgctcg tcgggcacga gcttcagacg ctccacgagc gcctggagct 11820 cctgggtgga ggggggccgg gccggcaggt agacgctgat ctcctcaatc ctttcgccgg 11880 ggcgggcagc cggtgcggga cggccggggc ggccgccggt ggggtgccgg gtcagtcgat 11940 gtagcccaga gcctggaggc gggccgccac ctgctgctcg tcgctcgcgg tgtacgccgg 12000 ctcctgcggg cgcggcgcgt agggggagaa ggcgcgcagt gccggggcca cctcgtgcgc 12060 gaacagttcc atggactcga tgcggtgccg gtccgccagc tcgccgaagg tgaactcgcc 12120 cagcagcgtg tcgaaatggc cctccaggga cgcggccctg atccggtcgg ccacggtcgc 12180 cggcgagccg acgaagacga ggttgttctc cagcaggaag cgcaggtcgc cggagcccgc 12240 caggatgtcc gtcgtgctga gcctgctggg tggggcgtgc tgatcgcggg aacgggacac 12300 cgagccgtac cgcagcgtct tggtgaacgt ctcggtgatg tgcgcggccg cccgctcctc 12360 cgcctcggcg tccgtccggg ccacgtggac cagcgtccag tagccgatgc gcgggtcgct 12420 cgcgtggccg tggccgcgcc accagtcgag gtagtgccgg tagaccagcg ccatggaggc 12480 gcgcggcacg atcatcgtgg aactggtgtg ggcaccggcc tcgctgagat agcgcaaggt 12540 gttgcggttg gtggtgggca cccacatcgg cgggtgcggg cgctgcaccg gcgggaagga 12600 cagggcgatg tcgcgcagcc ggtgcgcggg cccgtcgaag tcgaaggcgc cccgcgcggc 12660 gaacgcggca cgcagcagct ccagggcctc ccgggtcagg aggtgacggt tgtcgaaatc 12720 ggcgtcgaag ggcaggaagg ggtcacggct gacgcccgag gcgagcccca cctccagccg 12780 cccgcccagg agctggtcca gggtcgcgac ctcctcgacc aggtgcagcg ggtggcgcag 12840 cggcggcacc caccccatcg ggccgacccg gatgcgctcg gtgcgcagcg cggcgcccgt 12900 gcagaagacc gcgggcgagg gcatccagct ctcgtgcggc gtgcagtggt gctccaccga 12960 gaacgcgtag tcgaagccga gccggtcggc gtcctcgacc tcgcgccaca gctcttcgta 13020 gcgctcgccc ggcgtgatac cgggacgtcc ccagacatgg gagaaataag cgaatttcat 13080 tggcttcccc gggtgggcag gacacgtgga caactggacg tgctgggcgc cgcgtcgctc 13140 tgcggccggt gcaggggcga gggagggtcg acgccggcgg ccgaatagat gtggtcgatg 13200 caggaggcca ggatggtcac ttcggccatc gacccctcgc ccaggcccga caccgggccc 13260 tgcccgtcgg ccgagccgcc gagcctgcgg gcgagttcgt ccacctgccg ccggtactcc 13320 acgcccacgg gctcgtcggg cagcgcgacg gtctcctcca ccccctggcg caggaccacg 13380 aggctcgact tctgtagccg gtgggggctg aagccgaagg tgccgcgcag cgtcgccacc 13440 ccctcggtgc cctccacggt gatcgtggtg acgtccagcg cctggtggga ggcccagcgt 13500 gtctccaggg agatgcccac gtcggtgtcg gtgacgagga agccgcgggc ggtgtcctcc 13560 accgtctccc cggggcctgg ccgcgccgtg ccggaggagc gccggctcca gtcggccgtg 13620 gcctcgcccc ggctcatcca gtccgcggac atcgtgctcg ccgcccggac cacgcgcggc 13680 caccccagca ggtgcaggcc cacgtccagc aggtgccagc ccaggtcgag cagcgcgccg 13740 cccccggcga gccggcggtc gacgaaccac ccggtgcgct gcgggatgcc ggtggcccgg 13800 atccagctca gcccgacact gcgcacggtg cccagcgagg gcagcagctc cgccaggcgg 13860 cagacatcgg tgcggtgccg ggcggcgctc caggcgtaga gggtgatgtc cccgatgctg 13920 tcgccccgcg cctggtggtc cagggcgagc gcctgggcct cgaagagcgt gcggcacacc 13980 ggcttctcga cgaacaccgg cacgtcccgc tccaggaggg ccttggccac ggggagatgg 14040 aggtggttgg gcagggcgat gatggccgcg tccacgcttc tgggggcgag ctcttccggt 14100 ctgctcagga cgcgggtccg cgcgccctcc ggcagggccg acctggccgc cacggggtcg 14160 tcgtccacga ggaagtcgac ccggaacgcc gggtgttccg ccagcagcgg cagccacacc 14220 ttgcgcgaga cccatcccgc gcccagcact gccatccgga ggggttcgcg agctgtcgtc 14280 ggcggtgtca cgacgggtgc cttctccgtg aaagtcatca gaagcgggca ccaccgtcga 14340 cgacgagcgt attgccgttg acgtacgagg ccgacggcga cagcaggaac gacaccgccg 14400 ccgcgacctc ctccggctca ccgagccgcc ccgccccgat gaactgcagg accagatcgc 14460 gctcggcctc cgagaagtcc atcacctcgg cgcggatcgc cccgggcgcg acgacgttga 14520 cggtgatgcc gtgcttgccg acctcacccg ccaccgacgc cgcccacggc tggagcgccc 14580 ccttggtggc ggcgtacgcg ctgtggccgg gcagcccgct ggtcccggcc cgggagccga 14640 agagcacgat ccgcccgtac ctggcgcgca tcatcggctt caggcacgcc ttggcgagac 14700 ccacggaacc ggccaggttg acccgcagca gcttctccag gctccgggcg tccgtggcca 14760 tcgcgagccg gcgcgtacgc aagcccgcag cagccacaca gccgtccacc cgcccgaacc 14820 gttcgacggc cgccgccacc agcgcgtcgg cgccctcggg ttcgctcagg tccgccgcca 14880 cgggcacgag ggtgccgccc tggccctcca cctgctcccg cagcttgcgg atcgcctgct 14940 cgccgctgtg gtagccggcg acgacggtgg cgccgagcgc ggcgagctcc agcgcgcacg 15000 cgccgcctat ctgcccggac gcgcccgaga ccacgaccac gcggccgctc tgtcccaggc 15060 gctccgtggc ccggtcggtg acggtgctca tgaaccggcc tccttggcga tgatcagatg 15120 acagggggac gcgtccagcg gcacgtgccg tgcgacctcc gccccctctt gcagcagccc 15180 ggcgatcagc tcgccggtgg ccggccacgc ggtcccgccg tgcgtgagcc agtccagagc 15240 cagttcgctc cccggcccgg acgccggcag gaagacgtcg tcgaccagca gccgcccgcc 15300 cggccgcagg gagccgagca gggcaccgag agcgctgccc ggccccgggc cgtgcacggc 15360 gttggcgacc acgcagaagt cggcgtagcc gacgggcagt tccgtcccca cggtcaccct 15420 gccctcctcg accgccgcgg cgacggccga ggagagcggc ccgctcagcc ggccgacggt 15480 gaccagatgg ccgctcgccc cggggtccga ggcgagcagg cgttccagat agcggcccgg 15540 gcccgcggtc acctccacca cccgggctcc cggcccgggc cgcaggagcc gcaggccgag 15600 cgcggcccgg gcccgtgcgc cggggccgtc catcgcgccc tggtacaggg cgacgagcgg 15660 accgaggctc tcggggggac gctcctcgaa gggacgccgc gccgtcccgc tccgggccac 15720 cgcgacgagc tcctcgcgcg tgaccagccc acgggagagg tgctcctcca gcgccacgaa 15780 cgcggccagc tccccggccc ggacccggtc cccgggctct tgcgccccgg tggtcagcac 15840 ccccagagcg gtggccgtgc gcagcagcca ctccagggca tccgcgtcgc accccagctc 15900 cccggcgagg agagccgtgc cggcaccctg ggcgagtgcc tccagggcgc ccaagtcgtg 15960 cagcgcgaag agcacttcgg atgccttgta cgcccgggcg gcctccgccg cgctccgggt 16020 gaggcggtag acggacgccg cccgcacctt gcccgcggcg ttgaccggca ggctctcccg 16080 caggacgaac tcgtcgggca ccttgtgcgg ggccagctcc cggcgggcgt gctcgcgcag 16140 cgcctcgggg gtgagccccg gccccgccgc cgagacctcc gcgacgatcc cgtcctcgcc 16200 ccggtgccgc ccccgccggg cgcccacccg cacattcacc acgtccggat gaccgcgcag 16260 cacctcctcg atctccagcg gggagaccca gcgcccgccc cggcggatcg cccggtcctc 16320 gcgtcccagg atgcgcaggc ccccgggcac ggccacggcg agatcgccca tggcgtacgg 16380 ccggccgtcg acccgtacgc tcagcagccc cggggtgccg gcgggcggca ccacgccctc 16440 cgggccggtc agttcgcact ccacccccgg caggggagca ccggcgcaca agggctccag 16500 ccccgccggt ccggcgagca cggcgcccgt ctccgtggaa ccgtagttgc gggcgagacc 16560 ggtcccgaac gcctcggtga acgcgcggtc cagctgctcg tccaccggcc ccgcacccac 16620 catggccagc cggagaccgg gagcggcggg cgcccgcccg gccgctgctc cccgcagccg 16680 ccgggtcgcc agcagccggg ccacactggg caccagggcc accacggtcg caccaccgga 16740 cagctccgcg gcgatgcggc cgagggcggt cggcggtacg gggcgcagcg cggcacccgt 16800 cagcagtccg ccgaacagcc agcccagcgc gtacgcgtgg gacagcggca ccggcagcag 16860 cagggtgtcc tctcccgtca gcccgacccc gtcgcggtag cggcggccct ccgcgagcag 16920 gctctcctcg ctgcgggcga cgagcttgct cgcaccggtc gaccccgagg tcgggagcag 16980 cacggcgggc ggggcgccgg agggttctcc gggcgagccg gtcaacgtca ggcggaggcc 17040 gtcgccggtc ccggggacga ccagggacct gccgcccccg gcagcccgca gcagccgcgc 17100 ggtctcgggg ccgggggtgt cgggttcgag cagcaggggc ctggcgccgg atgccagcag 17160 ggagaggaag gcgacgaccc accgcgggct gttgggcgcg cgcagggcca ccgcctcgcc 17220 ctccaccgcc tcggccttga gctgtgccgc ggccgtacgc acctgctcca gcagggagtt 17280 cacatcggtc ccgggcagcg gtatccggcc ggacggcagc cgttccaccg cgcccaggag 17340 ggtgtccgcc tcgtgaccgg tcgcctgttc agtcatggcc gccctggagg tagtcggcct 17400 tcgcgtcgct gagcatctgc cggatgcggg gcggggagta ggtgggagcg gcgcacagct 17460 cgtcgagggt gatctcgtcc tcgaagtaca tcgagagcgt gtcccagggc gtgaagcagc 17520 cgtggcacgc cttgagccgg ggcttggggg cgagcagggc acggtagagg ccgctggtgc 17580 ccaccgtgtc cagcatctcg ctccagtcgt cctccagggc gttgcccatg tcggagaacc 17640 agatgttggg gcagggcgtg acgacgccgt cgctgaagct ggagacgacc agccgcggca 17700 gatggcagcg gaaggtgcgg cggccctcgc ggtagaagct cgtcagccgg tcgaagtagg 17760 gccgcggcgg gaggacccgc gcgaactcgt cgtagcggtc gacgagttcc tggatgtggc 17820 cgaactgccc gggccgcacc ttgaagtcct ccgagtccgg gccccgcacc gggaagggga 17880 agtagacggg aggccgggag aatcccgaca gccactcggc gaacgcgcag acctccgtga 17940 cgctccggtc gttgagcact gaatagatct ccaccggcag ccccgagtcc aggatccggg 18000 cgatggcggc gacgaccttc tcgtgcaggc tcccggacgg cacacgatgg ctgttgccgt 18060 ggtggaggtg gctgtcgagg gagacctgga gcacgacgtt gccccacgag cggaaccgct 18120 ccaggtgctc ctcgcgcacc aggacaccgt tggtctggat gaccagcacg tcgtatttac 18180 gggcctcctg ctccaggaag tccatgatcc cccggaccag gaagatctcg cctccggtca 18240 ccttgagcag cggcaggccg aagcggtccc ggatccggtc ggcgaccttg tccatgcgct 18300 gccccagccc gctgtccttg gcgtagctgt cgcgccgcgg gggctcgaag atcagttgaa 18360 gggagtggcc ctccttgagg ttgctctgtc cggtgaggca gtaggtgcag ctgaggttgc 18420 aggagtcctc gttgatgacc aggtcgttgc cgatcagcgg caggcgtcgc ctggtgcccg 18480 cgtcgggggt ggctgtcggc gggtgggtgc tacgggacat gagtggcctc tctcgtggtg 18540 gggctgcgca cggcgtgggt cacggccccc tttccacggg tgccggccgg gcctcgcccc 18600 ggtcgtcctg gccgacgggc acccagtggc ggtcggcgtg gccgggccgg ccgggcccgc 18660 cgtgtgcgcg ggcgcgggcg aacagggcct cgcgcagggc ggccgcaccg ccgaagtggg 18720 cgctcgcctg gctggagtag tgaccggcgg cggtcagcca gcggtccacc gcctcggacg 18780 gcagccgttc cgtgcgcggc cgcaggttcg tgtggtcacg ttcgcgcagc cggtagggga 18840 agtcctcgta gaagacggta cgggcggggg acaggggctc caccgcgccg cggaccaggc 18900 ggtggtcgac gtgccggccc gccgccaggg gaacgtggac gctcgacgcc cccgcgcaca 18960 gcggcagcag agccgcccgc acctcctcca gcagcgggag gtcggccggg tgccaggggc 19020 cgaagagccg gcgcggggaa gcgtagagat aggcgcccga ggccgtacgc agtgccgcgt 19080 cggtgaagcc cagcggcacg tggcgcacgc ccagttcggc acatgccgcc cggtcctcgg 19140 cccggcgcac cgcgggatcg gcggcgctcc gccacgactc gggcttcccg gccgcgggcc 19200 cggcgaagac cgtgacgacg gtcggccgcg ggccctcggc ggcccagcgc gccagccgcc 19260 cgcccaggga ccacacggcg tcgtccgcgt gcggggagag caccacggga ccgtacggcg 19320 cggtggccgg tgtgccgctc atcgcgccgc gtcccgtgcc ggtgcggcct cttcggccag 19380 gacggcgcgc acatacgcct ccggcgtgcc gtcgggcccg agcagggcct ccaaggcgcg 19440 taccgacggg tgcgggtggc cggcggagaa gtgcgtgagg ttgcggtggt gctgctccca 19500 gcggccgggg cgggcgtggc tgaggtgcac accgagggcg tcgggggcga gggtcctgcg 19560 cagcccggcg gcgtgcaggc ggaagccgaa ctccaggtcc tcgcaccccc aggtgagccc 19620 gaactcctcg tcgaatccgc cggtatgctc ccatgcggcc ttgtcgaggg cggtgttcgc 19680 gccgatgaag ccgagccagg gggcgacgtc cggcagggag ccgccggcca tggcctccac 19740 ggcccgctcc agggcgttgg cgacgagccg ccggtgcggt tggcgccgct cggaggcggc 19800 cggggcagcg ggttcgagtc cggcgcgggc gcggcggacc tcggtcgggg cggccttctc 19860 gacagcggcg aggaaccgcg ccgcggtggg gagttcgcgc agccggccgt gggtgaaggc 19920 gtccggttcc gcggccgcgg cgtgtgcggc gaggaagccg ggccccacca ggacgtcgtc 19980 gtcgaggaag accagccggg gcgcgagggc cgcggccgcc ccggcgttcc gggcggcggc 20040 ccgcccccgc agcggtcccc gcaccacgcg cagcgggaga aggccgctca tctcgcctgt 20100 cacggcgatc agttgatcac cggcgtcgcc cccgtcgttg tcgtcgacga cgaccacttc 20160 gaagggcggc gttcccgggg aggggccggc aaggcatgcg agggtcgcgc gcaggcgtgc 20220 cgcgggcccc cggctgggga cgacgacgct cagccggggg gcggtggtgc cgttgggggc 20280 ccgcatcggg tcagagcgcg ccgacgaggc cggagaagac ctccgccagc cggtccagcg 20340 tggtcacgtc ggcgagcagg acgcgatggt gcagccagag gcagtcgctg ccgatctcct 20400 ccgccacggg acagctcttg gccagctcct cggcgtccgc cggcgccggg ccgcgcgcga 20460 aaccctcggt gcggtagacc ggcgggaagc cgacgaacgc gggcactccc cgctcgacca 20520 gcgcgtccac cagcgcgagg cggcgccggg ccgagatgcc gggcagccgg accatggcca 20580 tgtagtggga gtggaggtcg ccgcgctcgt cgcgcccctg cggcaccacg ccgtcgatgg 20640 cggccagtgc cgtacgcagc tgggcccagc gctcctccct gatgcgcaac tgatccttca 20700 agcgcttcag ttgagcacgc aggacgctcg cggagaactc gttcatgcgg tagttggagc 20760 cctgcgtcag atggcggtag acgcggtccc cgggcgggcg gccgcagcag tgctggagga 20820 acgcctcgtg gaaggactcg tcgtccggca ggagcagggc gccgccctcg ccggcggtca 20880 tcagcttgcc gttctggaag ctgaaggcgg cgatcgagcc gagctccccg acccggcggc 20940 cctgccactg cgcgccgtgg gcgtgcgcgg cgtcctggag gaccggcacg cccgtcgcga 21000 cggagagctt ctccagggcg tccatgtcgg cgaactggcc cgccatgtgg accggcatga 21060 tcgctttggt gcgtggcgtc accagcgccg ccgccgcgtc ggcatcgagg cagtaggtgt 21120 cgggccgtac gtccgccggc accggcaccg cgcccatgcg ctgcacggcc agcgacgacg 21180 agatgaaggt gaacgcgggg acgatgacct cgtcaccggg gccgatcccc atgaccccca 21240 gggcgagttc cagggcgtgg gtgccgttcg tcgtggcgat cgcgtgcggg gcgccgtggt 21300 ggtcggcgaa ctcccgctcg aagagatcga cctcctgccc cgcgtcgcgc caccacccct 21360 tctggtccag ggcccgcagg agtccggcgc gctcctccgc gccgtgttgc ggccatgagg 21420 gaaaggacag gacgtcatca ccggacgtag gtgtcattga gcagcctttc ggtcctgcgg 21480 gtgcggcggc acggtcgact tcggggcgct gtacggcggg agggcgggtg tcgaggcctt 21540 tgccttcggt ggccgtggct tcacggcccg gttccgttgt gttcgccccg cgtcgggaag 21600 ggtggtgcgt gacccgacgg gaaacgccgt tcctcggggg cggcgggaaa tccggcccgc 21660 ggtgtgaggg gtggccggag cgggcatgtg atccggcccc cggtcatagg ccgggatcgg 21720 atgccagaca cgagcctcca tagggcagtt gccggagtca acacccttgc cgggaaggtc 21780 tgccccgacc gccggtcggc ggtggattct cgtcagcagg gtggttgagc agtgaaactg 21840 ccttattccc aagggaattg atccagttca ggggctgctc ggcgggcctg tcggcaacgt 21900 tatccggcgt cgaagtggct caaaccgcac ggctggaggg agcgggaagc gtcgcgtatg 21960 gtgggcgcga caccgtcctg gtatgtgctg tgtgcatggt tcattgagcc gaatcccact 22020 ccggccctcg gatccgggcg ccatacgatc accgttgtcc ggtctgtgga cgcaccggtg 22080 aggggctgtt acagtcctcg gatcatcgat gagcggcggc agtttctgcc tgcaatcgtg 22140 atgagttctc agagctggag gcaatttcgt gccaccctct ccccgcgccc tcgtcatcgg 22200 aatcgacgga ggcacattcg atacggtcga cccgctgatc gagtgcggtc tgctgcccca 22260 tatggcgaag ttgctgcgcg agagcgccag tgccgccacg gactgcacct ggcccgccca 22320 cacggcgccg gggtggagca cgttcgtctc cgccagcgat cccggcggtc acgggatcta 22380 tcagttctac gacacccagg acccggccta cggggcccgc gtcacgcgct ccggcgacct 22440 gggccggtcc tgcgcctggg actggctcgc cgcgcaggaa tattcgctgg gcctcatcaa 22500 catcccgatg tcgcacccgc cggccgacct ccccggctat caggtcacct ggccgctgga 22560 gcggacactc aagcactgcc gcccggattc cctgctgcgc gaactcgccg cggccaaggc 22620 ccatttccag tcggacctcg cgaccatgtt ccggggcgac atggcctatc tggaggaggc 22680 cgagcgcaat gtggcggcgc gggtccgctc cgtacggcat ctgatgagca cccggcccac 22740 cgatgtcgtg atggtcgtgc tcaccgaggc cgaccgggtc ggccaccact actggcacta 22800 cggcgacccc ggtcacccgg gccaccggcc cgccccggag ggcagcggct gggacgtcgc 22860 catgccccgg atctaccagg ccatcgacca cgcggtgggc gagctcctgg agctcgtgga 22920 cgaggacacc tccgtcgtgc tcgtctccga ccacggcctg ggcaccgggc gccacggcct 22980 gtcggtgcac accctcctgg aggaggccgg gctgctggcc accgcaccgg gggaggagcc 23040 gcaggacgcg gcggcgagct ggttcgcggg caacggccgg cacgtcgact tccgccgcac 23100 cagcgtctac atgcccgtcc ccggcagcta cggcctcaac atcaacgtac gcggacgcca 23160 gcagcgcggc accgtcgcac cccgcgaccg cgaacgcgtc atggacgagg tcacgggcct 23220 gctctccggg ctgaccggcc ccgagggaca gcaggtcttc cgggccgtcc gcccgcgcga 23280 agaggcgtac ccagggccgc acaccggccg ggcacccgac ctcctcctcg tcccgcggga 23340 cgagaccgtc ctgcccgtcc ccgacctcgg cggtgacgtg tggcggccga gcgcgcagac 23400 cggcctgcac cgctaccgcg gcctgtgggc gcaccgctcg ccccgcgtcc gccccggccg 23460 cctgcccggc accgtcgcgc tcaccgacac cctgcccacc ctgctcaccg acctcggggc 23520 cgcatggccc agcgacatcc acggccgccc cgtgaccgcc gtcctcgacg acggcgtacg 23580 cgtcccgccc tccgaccccc gggtcgaggc caccggcacc ccggccacca cgatcccggc 23640 cgccgcttcg gccgctgatg ccgccgagga cgcgtacacc agcgaccgct tgcgcgaaat 23700 gggctacctg taagcaccgc cgggccgtac cggcgcttgt ccccaccgga gtcccgccgc 23760 tcgcggcggc gtggaggaga gaggtatttc tgccatggag accctgacga ccgacaagat 23820 caaggaccgg ctgcgcaagg tgctcgtcga ttccctcgaa ctgtccctgg acccctcggc 23880 cgtacccgac gagggactcg tggagaagct gggcctggac tcgatcaaca ccatcgaatt 23940 cctcatctgg gtcgagagcg aattcggcat agagatcgcc gacgaggacc tgtcgatcaa 24000 gctcatcgac agtctcgacc tcctcgccgg ctatgtgtcc gagcgcgtga acggcgtcac 24060 cgcacccgcc gaatgacggc cgtgcgcgcg ctcgcctccg ggcccactcc ccgcagcgga 24120 aggacgtgag cacgatggac cggcacgccc tggtgatcgg gctcgacggc atgccgagga 24180 ccctgctgac ccgcctggcc ggcgacggga ccatgccgca caccgcggcg ctgctcgccg 24240 agggccactg cgcggaactg ctggcacccg taccggagat cagctccacc tcctgggcca 24300 ccttcctcac cggcaccaac ccgggccggc acggcatcta cggcttcacc gacctcgccc 24360 ccggcgacgg ctaccgcatc accttccccg gtgtgcggca gctgcgcgaa cccccgctgt 24420 gggaactcgc cgcccgcgcc ggccgcagga ccgtgtgcct gaacgtgccg ggcacctacc 24480 ccgcccccgc catcgacggc gtgctggtct ccggcttcgt cgcgcccgaa ctggagcgcg 24540 ccgtcagccc gccacggctg ctgccgctgc tgcgcggcct cgactacgaa ctcgacgtcg 24600 aggtcggcga cgtcgccgcc gacccggccg ccttcctcgg gcgggccgtc cgggccctgc 24660 gcgcccgcac ccgggcgatg gaacacctgc tgcgccagga gacctgggac ctcgcggtcg 24720 ccgtgctcac cgagaccgac cgcgtccacc acttcctgtg gcgcgcggtc gccgaccccg 24780 ccgaccccct ccacggggac gtcctcgcct tctaccgcct cgtggacgac tgcgtcgcca 24840 ccctggtgag caccctccca ccgggcggcg aactcttcct gatgagcgac cacggcttcg 24900 gacccgccgc ctgtcaggtc tatctgaacg cgtggctcag ggagtccggc tggctggccg 24960 ggctcgacgt ctgtccggac ctcaccgcgg tcgacgctcg cagcaccgcc ttcgcgctcg 25020 accccgcccg catccacctc aaccgcaaga gccgcttccc cggcggcggc ctgaccgacg 25080 cggaggcgga cgaggccgcc cacgagatcg cgcgcgagct gtccgccctg cgctgcgacg 25140 gcacccgcct gggccccgac gtcgacggac ccctgctcgt ccgcgacctc taccgcgctc 25200 aggagatcta ccacggcccg ctgttgggca acgcccccga cctggtggcc gtaccggccc 25260 ccggggtgca gctgcgcggc ggctggggcg gcacgcacac cgtacgcaac gacatcctca 25320 ccggcaccca cacccgcgac gacgcggtct tctaccggcg cggcgcgccc gcgcccgccc 25380 ccggggcgga cgacggcccc ctcgacatga cggacgtcgc cccgaccgtc ctcgcctccc 25440 tgggcatcca ccccggcggg ctcgacggcg cggccgtact cggcaccacg ggacccgcgt 25500 ccggtcacgg ccgcacggac ccccctctcg acatcaggga gctctgatga agcacgacct 25560 cggtctggca ccatcggcac ccaaaccggg aacactcgac ctgagcctgg acccacgcat 25620 cacggacccc gcttccttcc gggtcagttt cctgatcctc ctcgacggcg acctcgtgat 25680 gtcccccgaa cacctcggcg tcgcctacat ggccggtgtg ctgcgccata cgggcttcac 25740 cgcggagatc cgggaggtgg agcacggcga cgaccaggcg gccgccaccg tcgaggcgct 25800 caaggagtac cggcccgacc tcgtctgctt caccctgatg agcctgaacc tgggcagctg 25860 tctgaccctg tgccggatgc tgcgggagga gctgccgggg acgacgatcg cctgcggcgg 25920 cccagccggg accttcgcgg gcctggacgt cctgcggaac aacccctgga ccgacgtcgt 25980 cgccgtgggg gagggcgagc ccaccatcct cgacctcgtc caacggctct acctcaagga 26040 gccgttgtcc gcctgcaagg ggatctgcta ccgcgacgag gacggcacac cgcgccagaa 26100 ccccgcccgc cccctgatcc acaacctgga ggacctcccc ttccccgccc gggaccagct 26160 gcgccagcac ggcgacaagc tggagtacgt ccgggtcagc accagccggg gctgcgtcgc 26220 caactgcgcc ttctgctccg ccccgcacct gaagaaccgc gtccaggcgg gcaaggcgtg 26280 gcgcggccgc gggccggaac agatcgtgga cgaggtcgcc gagatcgtcg aacgccacca 26340 gttccggacc ttcgacttcg tcgactccac cttcgaggac cccgacggcg gccgggtcgg 26400 caagaaacgg gtcgccgcca tcgcgaacgg catcctggag cgcggcctcg acatctacta 26460 caacgtctgc atgcgggccg agaactggca cgacaccccc gaggaccacg ccctgctcga 26520 cctgctggtc gcctcgggcc tggagaaggt caacgtcggc atcgaggccg gcaccgccga 26580 ggaactgctc ctctgggaga agcgcgccac cgtcgaggac aacgtcacca tcatcaggat 26640 gctgcgggaa cacggcatct atctcgccat gggattcatt cccttccacc cctacgcgac 26700 cctggagacc atcgtcacca acgcggcctt cctgcgcgac aattccggcc acaacctccg 26760 gcgcatgacc gaacgcctgg agatctaccc cggaacggcc atcgtcagcc gcatgcgggc 26820 cgacggactc ctcggcgaga gctatctcga agggctcgac ccctacggct acgcattcaa 26880 ggatccccgc gtcggacggc tcgccaagca tttcgcccag ctctacaaca acgacgacta 26940 ccaccggcac ggcgtcatca ccgagcagtc ctccgtcttc gccttcgaga cctacaacgt 27000 cgtactccag accttcatct cccggctgca ccgccggttc accaccctgc cgggggtgga 27060 cgaggtgatg gaggcattca aggcccgggt gcacgagatc cgccaggaga tgggccggca 27120 caactacggc ttcttcatgt ccaatgtcga ggcggtcatg aacgacaccc tcgacccgga 27180 gaagcagcgc cggcaggtgg tggacgtcga gcacttcttc cgcgaccgcc tcgatgtgtt 27240 gcgcagcgag caattgcgcg tcggcaaggc cctcacccgg ctcggcgccc gggtgacgga 27300 ggtcagctcg accattccca aggagcgccc cggcggactg ccgcgccagt acacgggaga 27360 gggcagcggt gccacgtggt gagacgggaa ccgccgcggc gcgggtggcg gtctgcacgc 27420 tgagcagcag ggaactggtc ggcccgctgg cccggttgcc cggtgtggcg gccgcgggca 27480 cgctgatgac cgccaacctg ggcatcgagc aggtgatcaa ggccctgcgg tgcgaccgga 27540 cggtccgcgg cctgctcgtg tgcggccgcg actcaccccg cttccgcgcc ggccagagcc 27600 tgatcgccct cttccgccac ggcctgcgcc ccgaggacgg gcacatccgg ggagccaccg 27660 gctatctccc cgtcctgagg tcggtgacgg cgcgggagac cgaggaggta cgcgcccgcg 27720 tcgagctggt ggacgcccgt ggcgagcgcg acgtcgagac gctgcgcgcc gaggtcgcgg 27780 cactcctcgc ccgcgtacgg cgcaccccgg ccctcccctc ccgcgagcac gacggcggcc 27840 aacccagctt cgtggagccg gacttcggac ggctgcatcc tgtcggccgc cgccgctccc 27900 tggacgcggg catcggcggg ttcgtgctca tcagcgtcga ccgtgagcac cggcggatcc 27960 tgctgcgcca ctacacctcc gatgtgcggc cccggcacga gatgtggggc acccgcgggg 28020 aggcgatgct gctcgggctg ctggaggccg gcgtcatcga ggaccccgcc cacgccggat 28080 acctcggcgc cgaactggcc aaggccgaga cggcgctgcg gctcggcctg cactacgaac 28140 aggacctgcc cctgcgcccg ccgggcaggc cgcccggccc tgtgcggcgc cggaccgcga 28200 aggagcgaac gaccatggcg caagcacccg cgctggagga cttcctgcgt ctcgtgacga 28260 ggacgctggg ggccgaggac gccgtcctgg acctgcacac gccgctcggc gagcaactgg 28320 cggtggactc cgcccggctc atcgaactca ccgtcgtcct ggaggaggag ctcggcgcgg 28380 acctccccga cgacgccgac ctcgccaggg ccacccccgc ggaactccac aaagcactcg 28440 tgggctgagg aggagaccga catgcgcagc gtgctgttgc tcaacggacc caacctgggg 28500 acgctcggca agcggcaacc ggagatctac ggaaccgaca ccctggccga gatcgaggcc 28560 gccgtggccg aggaggtggg agcgcgcggc tgggaggtgg tctccgaaca gcgcaacggc 28620 gagggggaac tggtcgatgt gctccagcgc cacgacgacg tggtgggcgc cgtggtcaac 28680 cccggcgccc tgatgatcgc cggctggtca ctgcgcgacg cgctcgccga cttcgccccg 28740 ccctgggtgg aggtgcacct gagcaacgtg tggggacgcg aggcattccg gcacacctcc 28800 gtcacggccc cgctggcctc cggcgtcgtg atggggatgg gggcgctggg ctaccggctg 28860 gcagcgcgcg ccctcacccg gctggtcccc gaggactgac ggtgacccgg cccggcccgt 28920 acgcacctcc agatgggacc ggcccgcccg gcagggacgc cacctcggcg cccggcccgt 28980 acgcacgctc aggcgggcca cacccgcagc tcctccttga tcacctgagc gccggcctgg 29040 tcgcacgccc cgggcagcgg gcaggccgcc gggaggatcc gcacggtgaa cggcccctcg 29100 gtcaggccgc gccaggcggg gaccacggcg cgaccgcggt ccacctccgc ggacaccaag 29160 gccgtgaccg gacagggaaa ttgaccggag acctccccgc ccaccccttc gccgggcccg 29220 cggctgccga gccacagcag cacatgcacc ggcgggcgcg cggcccgctc ggcggcccgc 29280 agcgccgcgc ccagcccggc accggcacag accagcgccc acggcccgcc gtccccggcc 29340 gcctccgcga tcgcctcgac gccgtgctcc accaccagtt cgggggcgag cgcctgccgc 29400 cagccctccg cgtccggccc gtccgtcacg gcgaccagcc ggaccaccgg caccaccacg 29460 cctccggcgt tcccctcagc cgtacgcgac atccccagac cctctcttcc gtaccgtccc 29520 acccgccctc gctctcccgc ccggcgccgc tacggcaggc cgtcggtcat cccgagggag 29580 aagtagttct cgtaccccag cagccggcgc agttcgggcg tcgcgcgggt ggcgacgtcc 29640 tcccgcagac ccgagaagaa gttctgctgc tgccggacgt agccgcggca gtagtagttg 29700 aggatgccgt gccgcggccg gtcggtggtg ttggcgcccg tctggtgcca caggcgcccg 29760 tcgaagacca tcacgctccc ggccggcgcg cacacggcga ccgtctcggt gttcccctcg 29820 ccccggtcgt agtccggctg ccggcccagc agatgggagc cgggcaccag gcgggtcgcg 29880 ccgttgtcct cggtgaagtc gtccagcatc cacatgctgt tggccaccag cggatacggg 29940 ggccacggcg ggcgggcgaa ggtctggtcc gcgtgcagat gcatccggga accgccgggg 30000 cccgcgatat tggcgtgcgt gctggagagc aggaagccga agcccaggat ctcctccatc 30060 aggagcatga cggtgggatc ctgcacgttc tgctcgaatt cctcgccctt gttcagcagg 30120 ctgaagacgc gttggttgcc gccgtcgtag agaaaggccg agccgttctc acgctcctgc 30180 tcggcgacct ccagcagccg ccctctgagc ttttcgaaga ccgcggccgg caaggggcac 30240 tcgatcaggc agtatccggc ttcgaccaga tcccgggagg ctttctcgac gtcattcgtc 30300 aaagtcgcat ccatatggcg aggctagcag ccgaaatctc ggccgcacca tagcgcgaaa 30360 acgccggtcc atgatttttt cacgtgcggg aaggacggat tttccatggc acactcaccg 30420 cggcggccgg acggccccct ccgcatcggg gtctggctgg ccccccagca cacctcggtg 30480 gccgaactgc gcgccgcctg gcgcgcggcc gactccctgg gcgtggactc gctgtggctg 30540 tgggaccact tcttcccgct caccggggac cccgacggca gccacttcga ggcctggacc 30600 ctgctggcgg ccatggccgc cgacacccgc gccgcccgcc tgggcaccct ggtgtccaac 30660 tacgcctacc gcaaccccga cctcctggcc gacatggccc gcacggtcga ccacatcggc 30720 gacggccgcc tgatcctcgg catgggcgcc ggctgggtcg aacgcgacct gaaggagtac 30780 ggctacccca cgcccggcgc gggggagcgg gtggacgggc tcatcgaggc ggtggagcgc 30840 gtcgaccgca gactcggccg gctgcgcccc gggccgctcg gcgacctccc cctgctcatc 30900 ggcggggacg ggcagcggcg cctgctgcgc ttcgccgccg aacgggccgc catctggaac 30960 accatggcct ggcgcttcgc cgagggcaat cgcgtgctgg acgagtggtg cgcgcgggtc 31020 ggccgcgacc cggcggagat cgagcgcagc gccttcgtca cccgcgacca gaccgacgag 31080 gagctgcgct gcctggtggc gacgggcgtc cagcacctga tcttccaggt cgggcacccc 31140 ttccgcttcg acggcgtgga gcgggccctg cgcttcgcgg gcggctggag caaggggtaa 31200 ggccagggcc cggacgcgcc ccgcgtcgcc actagagcaa cgcgtccgcc agccggtcca 31260 cttgggacag cgccgccgcc gtggggtgga ggacgacctc gtccaccccg ccgtcggcga 31320 gcgccgagac cgccgcgcgg agctgccccg cggtgcgcgg ggtcttcgcc acgaactcct 31380 ccgcctcctc gcccagcacc gcgaagtagt cccggacgaa ggccgccgac tcctgggcca 31440 cgtcctcgcc cagggtgtag cgcgccagcg ccaccacatg cggcgccccg gcgcgtcccg 31500 cctcgctcca ggcgcggcgc acccgttccg cgaccggcac gatccgctcc ggctccaggc 31560 cgggcgccgt ccagccgtcg gcccagcgcg ccacgcggcg cacggccgcc gcgctgaccc 31620 cgccgacgag gaccggcaca ccggggccct ccgcgcccgg ccgggcgccc cggccgagca 31680 gctccagctg ctcctcgaac gccgcgcgcc ggtcgtcgaa ggcgcggccg gcggcctcga 31740 agtcgtcctc gcgcacgccg ggcccgaccc ccagggtgaa ccgcccgccc gacagcgagt 31800 ccagactcgc gaccgccttc gccagcacag gcgcggtgcg cagcgggccg atcaggacat 31860 tggtgagcag cccgatccgg gaggtcgccc cggccgccgc cgccagcgcc agcagcggat 31920 cgtggcccgg atacaccagg cgctcggtgg ccgcgagcga ggcgaatccc cgctcctcgg 31980 cccgccgcgc ccaatcggtt atcaggcgcc cgtccgcgcc gggcacggtg ttcggcagag 32040 caatgctgat cttcattggt ctccccgggg gttcgcagga tttccggtcg aatgtgacag 32100 gggattccgg cacggccggc gtgattgcgg caggagttca ccagcggccc ggcgcggaga 32160 aatgcggcgg catttccacg gccccctgtc ggaccgccgg accgccgtgt acgtttttcg 32220 gaaagcaacg tcgtacggtg cgcacagcga gaggaatccg cgatgcccgc tgccggaaaa 32280 gtcgccgtga taggactcga ctccgcgact ccgcagtaca tgttcgaccg gttcgccgag 32340 gacatgccgg tgttcaccgc cctcaggcgc aagtccctgt ggggtccgat gcgcagcatc 32400 gacccgccca tcaccatgcc cgcctggtcc tgcatgatgt ccggccgctc gcccggcgaa 32460 ctcggcgtct acggattccg cgaccgcggc gcctacgact acgggccgtt gaagttcgcc 32520 acctcccaca gcatccaagc cccccggatc tgggacgaga tgacggccgc cgggcgctcc 32580 agcgtggtcc tgggcgtccc cggcacctat cctcccgccc ccatccgcgg ggccatggtc 32640 tcctgcttcc tggctccctc cacacagtcg cgctacacct ccccgcccgg cctcgccgac 32700 gagctggaga agctcaccgg cggctacgcc ctggacgtgg aggacttccg ctccaccgac 32760 ctggaacgcg tatcccagcg cgtcttcgac atgagcgagc agcgcttcga ggtcgcgcgc 32820 cacctggcga ccacccagga gtgggacttc ctctccttcg tggacatggg ccccgaccgc 32880 ctccaccacg gcttctggaa atactgcgac cccgaccacc cgcgccacga gccgggcaac 32940 gcctacgccg gtctcttccg cgactactac cgcgccctcg accggcacct cggccgcttc 33000 ctggagagcc tgcccgagaa cacgaccgtc ctggtcgtct ccgaccacgg cgcccagccg 33060 atggtgggcg ggctcttcgt caacgagtgg ctgcgcaagg agggttacct cgtcctgacc 33120 gaggagcccg ccggacccac ccccgtcgcc caggccgccg tcgactggaa gcggaccacc 33180 gcctgggccg aaggcggcta ctacggacgg atcttcctca acgtcgaggg ccgggagccg 33240 cagggcacca tcccggccgc ggagtacgag agcacccgcg acctcatcgc ctccgccctg 33300 gaagcgctgc ccgacgacca ggggcagccg atgggcaccc gcgccctgcg ccccggcgag 33360 ctctacggag aggtcaacgg catcgccccc gacctcctgg tctacgtcgg caacctgcgc 33420 tggcgggccc tggccaccct cggcatgggc aagggcctct acacgacgga gaacgacacc 33480 ggccctgacc acgccaacca cggggacacc ggcatcttcg ccctcagcgc ccccggcatc 33540 acccccggcc gcgcggacgg cctgtcgctg tacgacgtgg cccccaccct gcgggaactg 33600 ctgggtctcg cgccgcaggg ctcccgcggc tccctcctcg gctgacatca cccgcccagc 33660 agcgcgtagg gagtgggcgg cgccggcacc ccgcccgctc ccgcaccgcc accgtgcacc 33720 acgtgcttgt ggcggtaggc gtccagctcg ttggtgagcc ggtcccagac ggcggagcgg 33780 ggcccggctg tgccgggcag ctccaggtcg accagccggt agtcgttgat ccatacccgg 33840 tccgcgcgca gccgctcggc caccgcgcgg gcccgcgcgg gatcggcgga ccacaccccg 33900 gcgctgagcc ggtagcggga gccgttggcg atgcgcaccg cgtcgtcgtc ggacccggcc 33960 cggacgaccg cgagcaccgg gccgaagatc tcctcctgcg cgacggcgtc gtccgcgccg 34020 accgacgcca gcaccgtggg caggaaatac gccccggcgt ccagcccggg cggcagctcg 34080 tccgccgcgg gcgcccggcc gccgcacacg agctccgcgc cctgggagag cccgagttcg 34140 gtgaagcgcc tggccgtacg cgcctggttg cgcgagacca gcggccccag gtcggtggcc 34200 gggtccagcg gatcaccgac gcgcagccgg cccacccgtt cgctcagcag ccgcaggaag 34260 tcgtcgtgga cgtcggcgtg caccaccgcg cgggtaccgg ccatgcacac ctgcccgttg 34320 tgcaggaacg ctccccacgt gacgccggtg accgcccggt ccagatcggc gtccgcgagc 34380 acgatgttgg gggacttgcc ccccaggtcc agccgggcgc tcgtccccgc cgcggcggca 34440 ccctcccgta cggcggcccc ggtctcgtcc gagccggtga acgccaccag gtcgacgccg 34500 ggcgagcgca ccagctgctc cccggcgacc ccgcccggcc ccgtgaccac gttgaccacg 34560 cccggcggca ggccgcactc gtggagcagc tccaccagtc gcagcgtgga gagcgaggcg 34620 aacgaagccg gtttgatcac acaggtgttg cccgcggcga tggcgggcgc gatgcgccag 34680 gccgccagca gcagcggcag attccacggc acgatcgcgg cgacgacccc caccggccgc 34740 cacacgacgt aggaacccga accgggcgcc tccggctgcc gttcgggcac gtgctccgcc 34800 caccacgcgc tccactcgaa ggctgccgcg gcccccggca catcggcccc gagagccttg 34860 cgcagcgtcg agccgttgtc gcgggcctcc aactcggcca gcggctccgc ttcttcacgc 34920 aagcgctgtg cggccttgcg cagcaggccc gcccgctcgc ccggcgccat ccgcggccac 34980 gggccctcgt cgaaggcccg ccgggcggcg gacaccgccc ggcggacgtc ctccgcgccg 35040 ccgctgggaa ggtcggccag gtggcgccgc gtggccggct cgaaggtgcg caggacggcg 35100 ccgtcgtggg cctgcacggc ctgcccgtcg atgtacatcg ggaaccgctc gaccgctctg 35160 tccacccggt ccatcgcctt caccttctcc ttctgctgac ccgtggggat gcgcccggcc 35220 gggcccgccc gcggccgcgg ccgtaccgga acacccgccc cggagcggcc gcgcccgcgg 35280 tcaggccggc aggggcggga tgttggggtt gaaccggaag acgttgcccg ggtcgtactg 35340 cgacttcagg gcctggagcc gcgcgtagtc ctccggcgtg taggcgctgc gggtcgtctc 35400 cctcgatgtg ttgtgacccg cgaggaagtt caggcacacc ccgggcgtcg tccacggccg 35460 catgctgtcg acgaactcct gctgcgccgc gtccacggcc gccagggtgt ccgggtcgac 35520 cagcgagccc acgtaggcgt tgaacaccgc ctccgggaag tggcccaccg cgctcgggtg 35580 ccggggcggc cgggcgaggg cgccgcccag gtgccgcagc tccaccccga acagcgcgtc 35640 cgtgcccggc cccgcgagcc tgaggatctc gtcgacggcg atctcgtcca gctgcccgaa 35700 catgaccgtt ttgctgtgac tcgacaccgg ggccggcgga tcgttgtgga tgatcccggc 35760 ccgcgtgtac gggagcgtgt ccaccgtatc catgacgacc gtgccggcgg cccggagctc 35820 ggcgaaccgg cgctcaccct cctcggggtc tcccagccag gccagccgga tgtgggtgac 35880 gaaccggccg cgcagcggtc cgggcacccc ctcggcatcg ggatacgcgg ccaggaacac 35940 cgacgacgcc atgtcctcgg gcatccgggg cgcccactgg agataggtgt tcagcacggc 36000 gcgcgtggag ccggcgtcga agaacagccc tccgccgtac acctgggtga cggggaacag 36060 cccgacctcg acggaggtga cgatgccgag gttccccctg ctgccgcgca cgccccagaa 36120 gagatcgggg tgttcctcgg cggagacctg gagaaaccgc ccgtcggccg tcaccaggtc 36180 gagcgagacg acatggtcgc cggcgaaccc gtacttccgc gacagaagcc cgagcccgcc 36240 gccgaggagg taggagaccg cgccgacgaa cggcgccgag ccgctcagcg gtgcaagacc 36300 gtgcgccgcc gcctcgtgga tcacctgctc ccagcgcacg cccgcctcga tccgggcggt 36360 ccgggcccgc gggtcgaccc tgacgccggt catccgccgg gtgctgatga ggacgtcggt 36420 ggccgccgag gacttcccgt gaccggtggc ctggacggcg atcccaaggc cccgggccct 36480 ggcgaagcgc acggcggcga tgacatccgc ggcaccggtg gcgacgacga cgagggcggg 36540 gcggtgttcc acggacagtt cgaagccgga gcgctcctcg tcgtacccct cgtccccggg 36600 caggaggacg gatccggcga cctgcgccgc gagctcttcg gcggccgcgg cgccgagggc 36660 cgcggacgtg tccgtcacgg agtggctggc tggtttcacc gaggaacctt tctggctgga 36720 gcttcgagaa gcgcgccgcg cgtgcgcggg cagggccgcg gggctcgccg gcccttggaa 36780 cggagcgggc cccgtcagtt gcgcgggccg gggaccaccg gcagtgaccg gacgcccagg 36840 ggcaggagtt cggagctgtg ctcgacctct tcgggaggca cggccagggc gatgcggggg 36900 aaggccgcca gcaggcggcc gatgccgacg gtcagttcgg tcacggccag cccggtcgcg 36960 gggcaggcgt gctgcccggc gccgaacgcg atgccgcggt cggccacccg ctcgatgtcg 37020 agcacgtggg gatcggcgaa gacctcgggg tcgcgggagg ccgccgagac cagtggcagc 37080 acggcgtccc cggcggcgac gcgcctgccg gagagcacca cgtcctcggt ggcgacccgc 37140 agcaggccgt cgttgctgga ggggtagtag cgcagcaact cctgtacggc agagggcagt 37200 acggaggggt cctcgcgcag ccgggccgcg aggccggggg aggagagcac gccgaagagg 37260 tgccgggcca gcaggtcgcg gatggtgatg aagcccgaga tgatcaggcc gtggagcagc 37320 aggcgccggt cgtcgtcggt gagctcctcc gcgtccagca gcgtgtcggt gacgctgtcg 37380 cccggctcgg ccctgcgggc cgcgagcagt tcgtccagca cctggccgag cctgccgcgg 37440 gcctccttca gcgcctgctc ggtggcaccg cgcggaagca gcagcagctc gacgtcggag 37500 gtgacgtcct gccaccggtc cccgggcagc ccgaggaact cggctgtgac gcggccggcg 37560 aagggcgcgg tgtacgccgc gacgaggtcc accgtgccgc tgccggcggg cagccggtcc 37620 agggccgcct cggcggccgc ctcgatgcgg ggtgcgaacc gcgccgtgcg cgaggcaccg 37680 aacgcggcca ccaccggacc gcgcagccgg ccgtgctccg gcgggtcgag gtccacgatg 37740 cccgagccct gggaccggcc gaagcccgag cccggcagca tggcggcgcg atggcggctg 37800 aagcggccgt cggtgagcac cgtgcgcacg tcctcgtgcc gggtcaccag ccagatgcgc 37860 gagccgtccg ccaggcgcac ctcggcgacc gggtcatcgg tgagcagccg cgcgtactcg 37920 ggcggcaccg tgccgccggg gccgggcggg aagggaaagg cgggcggggc ggctgaggtc 37980 atgcgccccg gctcctctca ccggccggcg cgccggcgcg ggcgtggccc ggccaggtga 38040 agtccttcgc caggacgcgg ttgtccagct ggtgttccac gacgatgttg ccgcagccgt 38100 agtcgtcggc gatgacctcg acgtcctcgg cggacaccag gtgtccgggg tcggccagca 38160 gggtcgccac gcccttggcg ggggccagga cctccagggc gaagccgcgg gccacgtgct 38220 ggcggtgggt gtgcgccgcc gcgatcagga aggtgtcgtc ggggtcgcag atcagatgcc 38280 acagcgcgcg ggcctggagg aagcggcgcg ggcggtcggt gaagcgccgg gaggagacca 38340 ggaccggccc gtccgcgccc ctgcgggtgc cgagggcgac caggccccgg tccatatagg 38400 ggacctgctc ggtgcggtag cccatcaggg gggccgggtc gaagggcgcg gtgtcgtcca 38460 gacccgccca cgcgcgcacc cggtgcgcca cctcgtagcc gaggtcccag gggttgtcga 38520 gtcgccccgg agcgaagaac ttctcgctca agtcggcgca gtccttgcgc agttccacca 38580 aggcgggagg gagcggggta tccgcgggcg cggtgcccat cagggtgcgg acgcgcgcga 38640 tccactggag ctggtcggct atccgctcag gccgcacccc gttgaagaag tcactggcga 38700 gcggttccgc caactgctcg gcggccttga ggatgtccgc ctcgtacggc tcggcctcgg 38760 cgtaggggtc caggcccagc cgtgccgcga tgcggcagaa ggcggcctcg tcctcgtcgg 38820 tggcgcggac ggcggcccac tcctcctgga gcggggtgcc ggtgatgccc tggtccgtga 38880 ggcgctcggt caccgcgtcg acgaacgagg ccagtgtggc ggtgaacgcg gcgctctcca 38940 cacaggagtt gccccggctc gcgaagcggt tcccgggtcg tacgtcgggg cccatgtccg 39000 gcatccatac gatccgggtc tcccggccct cgggcacgaa gagcatgtcc ggccagcgga 39060 agccgtcgca ggcggcgcgc aggatgtgac ggcgcgaacg catccaccac gaaccgcggt 39120 tgtcgcccac accgtggcgg taggcgaagc gcagctggga tatctgggtg ccgggccgcg 39180 cgtcggccac cagcgaccac cagttgaagg cgatccactc ggccaggggg tagagcgagc 39240 cggtcgtgtg ctcccggaag gtcccctgcc cgggctcctg gacgagtgtg acggtctcgg 39300 cgcccacggc gatgcgcagc cgggcccagg tcgcttgcag ctcgcctccg ccgccggccg 39360 gggcgtcgag ccaattccac tgcaattgga actcaggaag catggtccgc cagcccttcc 39420 ggccattcgc tcgggtggag ttcgtatccg gtgtattcgc ccggcgcacg gcccgtcagc 39480 cggaattcca cgacggagtc accggaccgg tgccagacat agcgcgggaa gccgtcctgc 39540 cagggaccgc cgaggaatcc ggtgcggatc cctgaacgga gggtgcccag cggggcggcc 39600 gacagggaac cgggcacggc gagcagatcg gccggggccg ggccgtattc ccggcgggcg 39660 aatgtcaccc gcccgcagag ttcttcccgt tcgctgcggt cgggcagtgg cgcgatcgcg 39720 cgacgtggct cgcggcggcg cccgggcgct cgtaggacca tgatgtccgc ctttcgggga 39780 acgtgccggt gagctgggcc ggcggggccc ggacgcggcg tgcgtccggg ccccgcccag 39840 ggtgttacgg gaggggcgcg aagaggtcca ccacgttgcc gtcggggtcc ttgacgatgg 39900 cgtagcgctg accccacacg gcgttccacg gcttgaggtg gccctcgtag ccggcgtcga 39960 cgagctcggc gtacttcttg tccacgctcg cggtgtcggg gaactcgaac gcgatggcga 40020 agcggtggcc gccggtgggg gcctgccact cggggtcgta gctgcgcacc gtctccacgg 40080 tgtcccaggc gagccggatg ccgccgtcga gcacggcctc cgtgtgcggc gcggagtcgg 40140 cctcggcggg gatctcgacg cccagcttcc ggtagaactc cagcgacttg gccatgtcct 40200 cgaccaccac ggcgaagagg gaaatccttg ctgacatgcg cgttcctttc ttgcactttt 40260 aaattggtct ccggtgccgg gccgtctgaa ttctccgggg ccggccggac cacgaagtcc 40320 gaatgtgctg gacgcgccgt acgctagtga ctgcgcgctg actttggcca atcggggtat 40380 cccccgccgg agtcaacgcc gctgacagga caacgatttc aggacagcgg cacgccgtcc 40440 cagtcgttcg gcacgtcacg cccgagcaga gcgcatacgg tcgcgggaat atcgacgggg 40500 gcggcggtgc gggtctgccg cgccccaccg gtgaagccgg ggccgacggc cgaccagtag 40560 gactcccggc ggtgccagcc gctctgccag tcgcggtgga cgtgcgaggc ccagtgcggg 40620 tcgcccagcg gaagatagcg ccagtccgcc ggctccagga tgaggtcggg ggcgtgctgg 40680 gtggcctcgc cgggatagac ctcctcccgg cggcgcaccg cgtcgaagaa cagcctgccg 40740 gtacgggggt tgcgccgctc cagcagcgcg gccgcgacgt cggtgcggac cttctcgtag 40800 tcgcgctccg ggaccaggcc gtgcttgtag cggtcgcgca ggttgatgtt caccccgtgc 40860 gtgccctgca ccgcctcgaa ggccgcgctg ccggcccact cgacgcggcc gtcctcggcg 40920 gtggccagga aacccgcctg ctccatctcg tcgttgatgg aacagtagtt gcgcagcggc 40980 ccgaagccta tctccgagaa ggccacgaca ctggtgcggt cgtcggccgc ccgcagggcg 41040 tcctggatga cctggtcgca ggtgcggtag gcggcgaaga cggcgctctc ccgctcgtgc 41100 tcggggccgt gctccagctc ctgccagtag atgtgcgaac agcggtcgat gctcgtgagg 41160 ttgacgatca cgacatcgga ctcctccagc agagccaatg ccgcgcgccc gcgctgcacg 41220 tccgcctcca gcagggaagg cagcagctcg tcgcggtcct gcccggtcca gaagatcgac 41280 acgtcgtgga ccggacggat gcccttcttc gccagggtgc gctggaggct gcgcgggtgg 41340 caggcgtgga gggtggcata catcggatag gtgatcaggg aaccgtcgaa gggctccggg 41400 ggatgggtgc cgaagaggcc tatcgaggcg aacctgacgc cctggaacac ctcgtgctgc 41460 cacagcagtg ggtggcggcg gtgctcgggg gtgaggacct gcggcgcgta ctccgggtcg 41520 tgacaggtcc agtaggagta gaagccgtgg tccgcggcgc gccggccggt caggacgctc 41580 agcaggcccg gcggttcgta gggggtgccc tcggcgtgga gcggcccgga agccccctgc 41640 gagcgcaggg cggcgaagcc gggcagcagc ccctgggcac accagcggtc gagcagctcg 41700 ggtgccgctc cctcggtgat gacgacgacg acacgctggc ggattgtcac gtgcgactcc 41760 ctcgggttgc gtggcagttg gcatgccgtc atccgggagg cgccggaaag gccgaggcgt 41820 tccggcgccg gacaggcgtc gatcgtcgga tcaagctaac agcgggacga ggactctctc 41880 cagacgacgg tacggaggaa attgagagag ggctgagaga gggctgagag agggcagagg 41940 cgggggagtg gcgtggggtc acacggtgcg caggaggcgc agacgttccc gtaccgcctt 42000 gggcagcccc gccaccacgc gatcgtacga atgctccacc atctcccgca gctcctccac 42060 gggaaccgtg ccgttcagga caaccgtgtt ccagtggcgc ttgttgacgt ggtagccggg 42120 caccaccgcc gcgtactgct cgcgcaggtg cagcgccaga tccggttcgc acttcagcgt 42180 gacctgcggc gggcggtcct cggaggcgtc ctggagaatg gcgaagacct tcttctccac 42240 cttgaagacc gcggctccgg ggccgaacgc ctcgtcgtcc accgcctccg gcagctccag 42300 cgcgaagtcg gagagttcct ctggtgtcat cgccggtcct tcttcctgcg gcacggcagc 42360 gagcggccga accgcgtggt catggggtcg gccaacagac tagaggcgca ggaggagttg 42420 ccgtgcggca gggcgcggac gctgatccac gatggccgaa acactgcggg gagttccggt 42480 cgcggcggga cggcgacctt gacgggcggt cctgccattg gcacagtttg gctggctcca 42540 cacaggtttt cggtggaccg ttcgttcctc tcccggtgct gcccggtcgc ggtaccggtg 42600 tccgcgcgat ccgtgtgccg cccgcgccgt cccgaaccgg cccgtgcgcc cactctcccg 42660 gccctccgcc gccggtctcc gtaccgccgc cccgcccttg ccggggcggc gccgacgccc 42720 gcaccccggc cttggccctg cccacggccg catccgcgca cccccctcac cccggcgccg 42780 gccatgcccc cgtgccgcct gccccccttg atgccctgtg gaggaacccc cgtatgaccg 42840 tggagcagac ccccgagaat cccgggaccg cggcccgcgc cgccgcggaa gagaccgtga 42900 acgacatcct gcaaggggcg tggaaggccc gcgccatcca cgtggccgtc gaactcggcg 42960 tcccggaact gctccaggag ggcccccgca ccgcgaccgc cctcgccgag gccaccggcg 43020 cccacgagca gaccctgcgc agactgctcc gactgctcgc cacggtgggc gtcttcgacg 43080 acctcggcca cgacgacctg ttcgcccaga acgccctctc cgccgtcctg ctgcccgacc 43140 ccgcgagccc ggtcgccacc gacgcgcgct tccaggcggc cccctggcac tggcgggcct 43200 gggaacagct cacgcacagc gtccgcaccg gtgaggcgtc ctttccttcg acgtggccaa 43260 cggcacctcg ttctggcagc tcacccacga gggaccccaa ggcgcgcgaa ctgttcaacc 43320 gcgccatggg gtcggtctcc ctcaccgagg ccggacaggt cgccgcggcc tacgacttct 43380 ccggcgccgc gaccgccgtg gacatcggcg gcggccgcgg cagcctcatg gcggccgtcc 43440 tcgacgcctt ccccggcctg cgcggaaccc tgctggagcg cccgcccgtc gccgaggagg 43500 cccgtgagct cctcaccggc cgcggcctcg cggaccggtg cgagatcctg cccggcgact 43560 tcttcgagac catccccgac ggcgccgacg tctacctcat caagcacgtg ctgcacgact 43620 gggacgacga cgacgtcgta cgcatcctcc gccggatcgc caccgccatg aagccggact 43680 cccggctcct ggtcatcgac aacctcatcg acgagcggcc cgccgcatcg acgctcttcg 43740 tcgacctgct gctgctcgtc ctcgtcggcg gcgccgaacg ctcggagagc gaattcgccg 43800 cgctgctgga gaagtcgggc ctgagggtgg agcgctcgct gccctgcggc gccggcccgg 43860 tgcgcatcgt cgagatccgc agggcctgaa accgcccctc ctgaccgaag ccggccacag 43920 ctgaaggagc aatgacacca tgacggtgct gggtctgggt ggatccggac atgactgggc 43980 ctcctgtgcc accgacggcc gacggctggt ggcgatcgac gaggagcggc tggtccgcag 44040 caagtacggc ctgggagcgg acctcctggc gggccacagc cggcgcgccg tcctcgacgc 44100 cctcggcacg agtgccgagg ccgtggaaca cgtggtggcc tgcgagctcg taccacgccc 44160 cttctaccac tcgttccgca ggcgcgtgac ggtcgtcaac caccatctcg cccacgccta 44220 cagcgcgttc ggggcctccg ggatgacccg cgccgccgta ctggtctgcg acaactccgg 44280 cagcctggtg acgggcctga agtccggccc agggccgcgc gaggcggaga cgatcagctg 44340 ctacaccgcc gacgcctccg ggctgcgcct ggtcaaccgg gtcgccggga cacacgccgt 44400 ggacgcctcc tccgagagcg cctactacca gcccggcgag accgacaatt ccctcggcca 44460 cttctaccgc tcggccagcc tcgcactcgg cctcgcctac tccggtccca agacccgcta 44520 ccccgtcagc gaggacggca agaccatggg cctcgcgccc tacggcgacg accgcttcgt 44580 cgacgaggtc gcggagctgg tcaccctgct gcccgagggc ggcgtgcaga tctcggcgag 44640 caaggtgaac cacctcttcg aacgcctcgt ggaatcgggt gagttcgagg accgggcggc 44700 cttggcctac gccgcccagg agacgctgga acgcgccctg ctgcactgcg cccgcgacct 44760 gcaccgccgc accggcctga cggacctgtg catcgccggc ggcgtcggcc tcaacagcgt 44820 cgccaacggc cggatcctgc gcgagacccc cttcgagcgg gtcttcgtcg tcccggccgc 44880 gggcgacaac gggatcagcc tcggctgcgc ctactacggc ctccacgagc tggaggggcg 44940 cgcgccgtcg gagctccccg ccctcgacac cgcctacctc gggcccgact accccgccga 45000 gcgcgtcgac gcggcgctgg ccggctcggg cttcaccgtg gagacccccg acgacctgcc 45060 cggcagggtc gccggcctgc tcgccgaagg gaagatcatc ggctggttcg acggccgctc 45120 cgaattcggc ccgcgcgcac tgggacaccg cagcatcctc gccgcaccct tccccgcctc 45180 cgtgcgggac cacctcaacg acaacgtcaa acaccgcgag tggttccgcc cctacgcccc 45240 catcgtccgc gaggaccggg cggcggacta cttcgacctc gtccagccct ccccgttcat 45300 gctggtcgtc gcgcgcgtga cccggcagga cgccatcccc gccgccaccc acgtggacgg 45360 caccgcccgg ctccagacgc tgaacgccgc acagaacccg aaggtctacg agctgctcgg 45420 caggttcgag gcgctcaccg gctgcgccgt gctgctcaac acctccttca acgtcgccgg 45480 ccagcccatc gtcgagaccc cggaggacgc cgtcgaggcg ttcgcgggca tgcgcctgga 45540 ccacctcgtc gtgggggacc ggctggcgac caagccctga cagcacgccg aggcccgcga 45600 ccggcaggga ggagagccaa gcggtggacg tccccgtgct cgtggtcgga ggaggaccga 45660 cgggcttggc gatggcgctc ttcctcgcac gccacggcgt cggctgcctg ctggtcgaac 45720 ggcggacgac cacctcgccc gtcccgcgcg ccacccacgt cagccgccgc tccatggaac 45780 tcttccgcga ggcgggcctg gaggaggaga tccgccgggc cgggttcgag gtcgtgcgcg 45840 aggacgaccc acggctgcgg acccggcccg aacgccacct gccccgggtg gtcctgcaag 45900 ccgcctcgct cgccggcccc ggcccggtgg gggtcctgga gaccggtgac gaggaactgg 45960 ccgtacccgg cccctgcgca cccttctggt gcggccagga ccggatggaa cccctgctcg 46020 ccaaggccgc ggcgcgccac ggcgccgatg tgcgcttcgg ccacgaactg accggcctgt 46080 ggccggggga ggacagcaca cgggcccgcg tccgggcagc gggaacggga cggacctaca 46140 ccgtcgacgc ccgcttcgtc atcgccgccg acggggcgcg cggcgagatc gccgagcgcg 46200 tgggcatcgc gcgggagggc ctgggcacgg tcgcccaccg ggtgagcatc ctcttccgcg 46260 ccgacccggg gcgctgggcc cgcgaccggc ggttcttcat gtgcatgatc cagaacccgg 46320 ggttcgacgg ggcggtgatg gagctcaaca ccccgggccg ctggtgcgcc gcggtggact 46380 acgacccggc ccgcgccgaa cccgacggca cctactccgc acgcacctgc ctcgacctgg 46440 tccgggccgc cgtcggtgac gaccggagcg acgcggcggt cgacaccgtc ttccactgga 46500 aggcccggca ccgcatagcg gccgcctacc gcagtggggc ggtgttcctc atcggcgacg 46560 ccgcccacct ccacccgccc tccggcggct acggatccaa cgtcggcttc caggacgcgc 46620 acaacctcgc ctggaagatc gccgccgtgc tcggcggctg ggccggaccg cggctgctgg 46680 acacctacga cgaagagcgc cgccccgtgg gaaaggcgac ggcggagcag tcgatgctcc 46740 tcgacggcgt gccaccggaa ccactggggg gaagcgtcgt ccgctgcgat ccccgcaccc 46800 tgatcatggg ataccgctac cactccgccg ccgtcctcgg ccccccgcac ggccccgcct 46860 tccccgcggc cttcaccctg cgcggagacc cgggcacccg gctgccgcac gtatggctgc 46920 gtacggacgc gggggaacgc gtctccacgc tcgacctgtg ccacgggcac ttcgtcctgc 46980 tctccgccga cccggtctgg gcggcggccg cggcgcgctc ggcgaaggag acgggcgtac 47040 cgctgcgggg ccaccacctg gcggccaccg gaagcgaact cgccgacccc tccggcgagt 47100 tcccgcggag ctgcgggacc gggcccgcgg gggccgtgct cgtacggccg gacggcatgg 47160 tcgcctggcg cacggcccgc gccgtgcccc cggacccgga cagcgcgcag gacctggtca 47220 cggcagcggt gagacgtgtc ctcgcactgc cggagcgcgc ggcgccaccg gtgctcggtc 47280 cgccgcggtt gtcacgcggt tcctatcggc gagtcgggag cgacgggtga agcctcattc 47340 cttctgcacg tgctggccgg gcgccaccgt atggctgacg ggcccaccgg gcgcgggcaa 47400 gacgacgatc gcccgcgcac tggcggagcg gctgcgcgaa cggggccggc gcgtggaggt 47460 gctcgacggc gacgcgaccc gcgcgctcct gaccgcgggc tcctcgtggg aggaccgtgg 47520 caccggcctc cagcgggtcg gcctgatggc cgaggtcctg gcgcgcaacg gcatcgtcgt 47580 cctcgtcccg gtgaccgcgg cccgcgcgga cagccgcgaa gccgtacgca gacgccacga 47640 gcggtccggc accgcgcacc tggaagtgcg ggtggtccgg gacgcagtgc ctccgagcgg 47700 gctccccgcg ccgcccggcc cagatctgcg gatcgcggcg cacgagcaga gcgccgagga 47760 gtcggcgcgg gcactgcacc ggctcctggc ggagagggag ctggcgtgaa ccccgggcgc 47820 ggtggagcgt acgccgcggg gcgcgacggg acccgcggga cgcgacgccc tcacggtctg 47880 tcgcacctgg atctgctgga gtcggagtcg gtccacatct tccgtgaggt ggcgggcgag 47940 ttcgagcggc cggtgatcct cttctccggc ggcaaggact cgatcgtcat gctgcacctg 48000 gcgctgaagt ccttcgctcc cgcacccgtg ccgttcgcgc tgctgcacgt ggacaccggc 48060 cacaacttcc ccgaggtgat cgcctaccgg gaccgcgtcg tggcggcgct cggtctgcgg 48120 ctggaagtgg cctccgtgca ggacttcatc gacaacggca ccttgcgcga acgcccggac 48180 ggcacccgca atccgctgca gacggtgcca ctgctggacg cgatcgggcg ccaccgcttc 48240 gacgccgtct tcggcggcgg ccgccgcgac gaggagaagg cccgcgcgaa ggagcgggtg 48300 ttctccctgc gcgacgagtt cggcggctgg gacccgcgcc gccagcgccc cgaactgtgg 48360 cggctctaca acggccgcca cgcacccggc gagcacgtcc gcgtcttccc cctctccaac 48420 tggaccgagc tcgacgtgtg gcagtacgtc gcccgcgagg agatcgaact ccccaccatc 48480 tactacgccc acgagcgcga ggtcttccgc cgcggcggca tgtggctggc accgggggag 48540 tggggcggcc cacgcgaggg ggaagcggtg gagaagcgac gggtgcgcta ccgcacggtg 48600 ggggacatgt cctgcaccgg cgcggtggac tcggcggcgg ccaccgtggc cgacgtcgtc 48660 gccgagatcg ccacgtcccg cctcacggaa cggggcgcga cccgggccga cgacaagctg 48720 tcggaagccg cgatggagga ccgcaagcgc gaggggtatt tctagcgcgg cggggccggt 48780 gcggcccaca agcggaggac tagtccctaa gtatgaagtc ccctactccg tttgtctgtt 48840 gagggcaggg gcgccgtctg aggatgatgc agtccatgtc acagttactt tccgggaagg 48900 acggcgccca ggaggcgcca agtcgcggcg ggtccacgtg ggtggcggtc ctcgccgcgt 48960 gcgtggggca gttcgtggtg gtcctcgacg tgtccgtcat caatgtcgcg ctgccgtcga 49020 tccgttccgg cctcgacatc ggcgagacgg gcctgcagtg ggtggtcaac gcctacgtca 49080 tcgccttcgc gggcttcctg ctgctcggcg gccgggcctc cgacctcttc ggccgcaagg 49140 ccgtgttcgt cttcggcctc ggggtgttca ccgccgcgag cctgctcggc ggcctcgcgc 49200 aggcgccgtg gatgctcatc gtcgcccgcg ccctgcaagg catcggggcg gccgtgctct 49260 cacccgccac cctcgcgatc ctcaccacca cgttccccga gggtccggcg cgcatcaaag 49320 ccgtcgcgat ctggacggcc gtgggcacgg gcggcggcgc ggccggcggc ctcatcggcg 49380 gcctgctcac cgactacctc tcgtggcgct gggtgttgct gatcaacgtg ccgctgggcc 49440 ttgtcgtgat cgtcgcgacc gtcgcctggc tggccgagag ccgcagcgac caggcacacc 49500 gacgccggct ggacctcccg ggagcggtgc tggtgaccct gggcgtcggc agcctggcct 49560 acggcatctc gcagagcgag ggccacggct ggggctcgcc gcggacgctc accttcctga 49620 tcgtcggtgt cgtggcgctc ctcgccttcg tcgccgtgga gcagcgcacg cgcgagccgt 49680 tgatgccgct cggtgtcttc cgggtgcgct cggtgtcggc ggccaacgcc atcaccatcg 49740 tcagtggcat gggcttctac gcgatgtggt acttcctctc gctctacatg cagaacgtgc 49800 tgaaatactc cgccgtacag accggcctgg ccctgcttcc ccacaccgcc accatcatcc 49860 tctccgcgca gttcgcaccc cgcctgatgc ggtggatcaa ggggcgcacc ctcctcgtga 49920 tcgcgggact gctgaccgcc gcgggcttca tctggcaggg gaacatggac gccgacggct 49980 ccttcctggc gaccctgctc ggcccgggaa tcgtcttctc cttcggcgcg ggcctgatga 50040 tgacgctcct cgcggtctcc gccacgacgg gcgtggagct ctccgaatcg ggcctggtgg 50100 ccggcctcgc caacacctcg cgcaccatgg gcggcgcgct cggcctgtcg gtcctcgcgt 50160 ccgtcgccgc ccgccgcacg gccgacgtgg ggcccggcgc ggagggcctg gcctccggct 50220 acggtcgggc gttcgtcgtg tccggggcca tcatcctcgt gagcatgctg atgatcccct 50280 tcctgcccaa gccccagccc cagaccccgg cggaatgacc tgtgagcacg gacatacgag 50340 gaggcttcgt ggggcaggac agccggccgc ggtggctcac cgacgaggaa caacgcgtgt 50400 ggcgcggcta tctgcgggcc accaggctgg tggaggacca cctggaccgc cgcctccagc 50460 gggaagcgga catgccgcac ctctattacg gtcttctcgt ccagctctcc gaggccccgc 50520 gccgggggat ccggatgacc gaccttgccc gcaacgcgaa gatcacccgc ccgcggctct 50580 cgcacgcgat cacccgcctg gagaagctcg gctgggtgcg ccgggaatcg tgccacggcg 50640 acaggcgcgg ccagaacgcc gtcctcacgg aagagggccg cgaggttctg gagaagtcgg 50700 cgccgggcca tgtcgccgct gtgcgcgcgg ccgtcttcga cagcctcacc ccggaacagg 50760 tcgggcaact gggccggatc tgccaggcga tagagaaggg gctggaccgg gaaggcgcgg 50820 acctgccgtg gctgcgctga ggcgggaagc cgtcgcgagc gcgcggggcc gtcaggctct 50880 gacggccccc gccgcccgcg tacgggatcg ggccgaccgc gccccggatt cacgcgagtc 50940 cgggagcaga ccggacgaca cggatattct ggatgccgtg gaacgacacg acggggcacc 51000 gggctggggc ttcacccata cccagtacag cgcggaccac ggtgaacgcg gcgccacccg 51060 cagggccggg gccctgctct ccgcgcggcc cctgccgcag aaccagcaca tcatgggctg 51120 gggcgcggag aatcccgaac cggcgcccgg acgctacgac ttcgaggtcc tcgacgagcg 51180 cgtcgccctg atgcgcgcga cgggggccac gcccgtcctg accctgtgtg ccgcccccga 51240 ctggatgaag ggcggccggc ccggccgcac cgactggtcg cgactggaga ccgcccccga 51300 cccccggcac tacgcggact tcgcccggct cgcgggcgtg atcgcccaac gctacccgga 51360 catcaggcac ttcctcgtgt ggaacgagct gaagggcttc tacgacgagg acaggcggcg 51420 ctgggattat gagggataca cccggctgta caacctcgtc cacgccgagc tgaagcggcg 51480 gaacccgcgc aatctggtgg gcggccccta tgcggtggtc gaccacgacc cgcccgccga 51540 ggacgcggcg gaccgctcgc gcgaactgcg cggtccctgg ggcgagctgg accagcgctc 51600 cgccgacgtc atccgctatt ggaacgccca caaggcgggc gcggacttcg tcgtcgtcga 51660 cgggtccagc tacacccgcg agggccaccg ggcgattccg gacgagttcg ccgccaccga 51720 gaagttcgcc gacgtcaccc gctgggtcag gagcgtgacc ggactcccgg tgtggtgggc 51780 cgagtggtac gtcgagccgc ccgccgagga cgaccggccg ggcggccggg acggctgggg 51840 cgaggggcac cgcaccgccg tgcaggccac cgcgatgatg cggctggcgg agagcggcgc 51900 gtcggccgcc ttctactgga acccgcagcg gaccgggaag gcgtgccccg gctgcctgtg 51960 gcggagcacc cacttgcgcg acgggggagg ggagttgccc atggcgggtc tcctgagccg 52020 gttcgctcgc gaattccctc cgggcaccgc cttccggccg gtcgccgtca cctgcgggag 52080 cggtgacagg gtcgaggccc tcgccgacga ggccgccgtg ctcgtcgtca acaccgagtg 52140 ccggccggtg gccgccaggg tggacgggca ggcgctgtcc ctcgcgccgt acgaggtgcg 52200 ctggctgacc cgcccgtaat ccagtggggc ggcgcacggg cgcggacagg gaattgcgga 52260 acagggaagt tcacgaataa ggagaacgcg ggaaagcgct cgggcggagc gtgaaacccc 52320 tgtcggcgct cacgatatcc acccagctga tttgcaggtg aaacgggcgg tcgcctcgac 52380 ggtgccgccc gtttcctgtt gcccgaaagg gcaatcgggc atcagcagga gagattgccg 52440 cccggcgcca cgccgaggat cttggtgaat cgctggtagt tcgcgacgcg gctctggacc 52500 tgggcggggt tgtggccgtc gcactccagg gcgccgttga tgctgcggat ggtctgcccg 52560 aagccgcggt ggttgaccat ggcctcgtgc ggggtcatgg tgccggggcc gcgctgggtg 52620 ttccagtacc acaggccggt cttccaggag acggccgcgt ccttctgcac cagcgagggg 52680 ttgtggagca ggtcgatgcc gagggcgtca cccgccgcct tgtagttgaa gttccagctg 52740 atctggagcg ggccgcgacc gtagtaggcg gcctggcctg ccggacagcc gtagggccgg 52800 ctccggtcgc agtagtgggg gtagttggcg gtgttctgct ccacgacata gaccagtccg 52860 ccggtctcgt gggcgacgtt ggcgaggaag gcggcggcct cctgcttccg gacctcggcg 52920 ctgccggtgc ccgcgaagcc cgggtacgcc ttgagcgcgg cgaccaggcc cttgtacgta 52980 tagaacgcgt tccgcttcgg gaacatctgc ttgaactggg cctcgctcac ggggaatgcg 53040 gcggcctgcg aggtgccgcc gtgcggtgcg gccgcgctcg ccgtggtggc gggggcgagg 53100 acggatatgc cgaccagtgc cagagcggcc ggcagcaggg cggcgatgcg atttcttctc 53160 atggcggctc ccgtggggga aagggtgagt gacgcccgcc gacggtgaat cgggcccgtt 53220 gggcgccttc gcgtcatcgc gcagtgaata actcccgtga gtttggtgtc aatggcatgc 53280 gccgtgtccg gccgaaccag gtgcactgag caatgagttc aggacaactg cggccgatag 53340 ggcttgcggg agcaacgagg accatgacct catatgccgg aagccggaca cgtgccgaga 53400 aatgccgctg tcctgtggct ccttgggtga cctgtgaaac ccggctggct catgaacgag 53460 ccgattgaac gagccgattg aacaagccga tgaacaagga 53500 77 77 000 78 78 000 79 621 DNA Streptomyces lavendulae 79 gtgccccgga atctcctgga cgtcagggac gtccaccaca cctacggcac ccgcagggtg 60 ctgcgcggca tcgacttgtc cctgcgcccc ggaacgctgg ccggcgtcgt gggcgagaac 120 ggcgcgggga agtcgacgct cctgaagatc ctcgccggtg agctgcggcc gcagcgcggg 180 caggtccact acggcggccg gttcggttac tgcccgcagc atctcgtcct gaaccaggct 240 ctcaccgtcc gccagcacct ggagtatttc cgggtggcgt acggcctcgc caccctcagc 300 catgccgaga gaatcatgga cgtgctccgg ctgtccgact acggggacga gcgggtcagc 360 gtgctcagcg gcggcacgaa acagaagctc aacctcacac tggccctgat gcacgaccct 420 gacctgctcc tcctggacga gccctaccag ggcttcgact gggacaccca ccagcggttc 480 tggagcctgg ccgccggtct gcgcgaacgc gggcggtccg tgctggtcgt ctcccacctg 540 gcctacgacg ccgaacggct cgacgaactc cggcacctcg acggcggact gctgcactca 600 aggacgacgg caccggtatg a 621 80 1350 DNA Streptomyces lavendulae 80 atgaccggcg ccctcctctg tcccagggac atctccccga ctttcgcggc gaacgcggac 60 ttcatccggc agcgcatcga ccgcacggcc gaacgcatca accatcacat cgaccgcctg 120 tgcccgaacg cgtcggcaga tgcttgctcc acgtggctgc caccgggcca catcaccggg 180 acgaccgggc ctggcacgcc gccggtcgtc gcccagcggc tgcaccgggc cctgacctct 240 cccgtccgcc atctgaccga tgccggagga cagcgctggc ggccggtgct ggcctgggag 300 gccatcggtc tgatgggagg tgacagcgaa tcctgcggcc tgctgatcgc ggcgagtgag 360 ctgctccaca ccggatccct catcgtcgac gacgtccagg acgcctcacc gctgcgccgc 420 ggacaaccgg ccgtgcacac catgttcggc atgccgactg cggtgaacgc gggtacggcc 480 gcctatttcc tctgggagcg ggccgttcag ctcacctttc ccgacgacgc ctcgcggtgc 540 ggggagttgc gggcactggg tctggccgcg ctgcgagcgg ctcacgccgg tcaggcactg 600 gatctccaag gtcaccggga agagatggac caggccgtgg ccggcgacga ccggcacact 660 gtgctggaac tggtccgtct gacacaccgg ttgaagtccg gggccccggt ctcggcggcc 720 atggaggcag caggggtcgt cacgggtgcc gagccggaac tgcggagagc actgggggct 780 ttcggttcag cggtgggcac cgcctaccag atcgccgacg acgtcgccga cctgagcggt 840 gtcacacggg cgggggcacc gacgaagcag gccaccgagg acctgcggag cggcaaggtc 900 accatgccac tggcccacgc cgtggtccgg ctgccgcaga gcccggctga accagctctg 960 gcagcaggtc aaggacggct cgggcagcgc gacggcggtg gccgaggtgt gccgggacct 1020 gaacgcctgc ggagcggtga gagcctgcct ggaggaagcc gaccagttgg tgagcgacgc 1080 gtggaacaag ctcgagcccc tgctgccgtc ggcaggccac agccacttgt tgtacgagct 1140 ggccctctcg gtcgtccggc gcgaccaggt cgcctgagga gaggaagccg tgccccggaa 1200 tctcctggac gtcagggacg tccaccacac ctacggcacc cgcagggtgc tgcgcggcat 1260 cgacttgtcc ctgcgccccg gaacgctggc cggcgtcgtg ggcgagaacg gcgcggggaa 1320 gtcgacgctc ctgaagatcc tcgccggtga 1350 81 1425 DNA Streptomyces lavendulae 81 atgcgggcgg gaggacgcct gatgaacgga gattcgaagg cggactccgt tgtgccgatg 60 gctcctgact gcagtcggtt gctcgggcat gcccgctccg tgcggttctt gatggatctg 120 agggagatcg cggcccggct cggcaaggcg gggcctgtgg tcagggtgaa ggcgggcccg 180 ttcgtcgggt acctgctcaa tgactcctcg ctgatccggc aggctgctcg cgacgaggac 240 accttcatgt tctggggcag ggaaccccat gtgcgggtga tcgtaagaga gggactgctg 300 agcaccgagg gaacggtcca ccgtgaccgg cgggcggtga tgaggcccgc gttcgccgtg 360 ccccggccgg cggacctcgg ggcgtccgtg cggaccgaga cccgaagtct gctcgccacc 420 cttcccgcgg accgtccggt cgacatgagc cacgagatga cccggctcac tttccatctc 480 gccgtcaggt gcgtcttgcg cagcgaggtc tcgcccggaa cgctgaccgc gctggccgcg 540 gcgcatgcca cgctgtcgca ggtcggggcc ttgcggtttc tcctgtcccc gtggccttgg 600 gtacccgtgc caagacaacg cgcgctccgc cgtgccctcg cggtgctgga cgaggccacc 660 cgggaagtcc tcgcgcgcca tcggccggct gaggacgggt gtgatgtggt ctccctgctg 720 aagcaagcgt ggcgcgagcc ctcggaagcg gcggtgcagg acgtgcgcac gctgctgttc 780 accggcgggg aggccaccgc ctcgacactc gcgtgggcct gctacgaact gggccgccac 840 ccccaccatc aacaggcctt gcaggaggag gccgacgcgg cactcggcct ggggcactcg 900 caggccggcg tcggaataaa ccggttgccc cggacagccg ccttcgtcaa ggaagtcatc 960 cggcttcacg gcctcccggt ccttgtgcgt tgcacgcgcc gcgagacgtg gctgggcggc 1020 caccggctgc ccgcgaaggc gacggtgttc ctccacctcg gggcgatgag ccgtgacccc 1080 gcccacttcg aacggcctga tgtcttcgat ccgacgcgct ggatgccgga cgctcagccc 1140 tcggtctccc ctgctgcctg gcttccctat gcgctcgggc cccgctactg ccctggtgcc 1200 gcggtggccg acgtcatggt gcccgtcgcg ctcgccactc tggtcgccac gcggaccgta 1260 cgcacggcgc gtccgggccg gacggtgcgc tccggcttcg aactcgccgc gatgccacgc 1320 ggcctgacca tgatcgccgc actccgcgag ccgtgcccga gcagtccctc cgcggcatcg 1380 ggccggcgac cccatgaggg agcctcgtcc atcgcccgcc cctga 1425 82 1821 DNA Streptomyces lavendulae 82 atgaagatct ctcgaatagg ccgcgcgtca tccatcgccg ccctggtgac aaccgcactc 60 gctttcacgg cagttggcac cgtcgctccc acggccgtcg ccgactcccg cgcggccgcc 120 gcttccggga cgcagaatga ccacccgagc tcggggcagg gcacctccac ctctgagctc 180 cggcgcaagg gcctggtccc gtcgagtctc gtggccaagc ccatcacccg cagcgagacc 240 ctcagacgcg ccgccagctg gttcggcaag ggtctccact acagcgggga caacacctat 300 cagggctggc gcacggactg ctccggcttc gtctccatgg cctggggact gcccggcccg 360 ggtgagacca ccgattcgtt cattcccggg ggcgtggccc acgaaatctc caaggacgaa 420 ctgaagcccg gcgacgcgct caacaacaag gcgctcggca acgacggtca cgtcgtcctg 480 ttcgagaagt gggccgattc ctcccagtcc tcctactggg gttatgagtt cagcagcagc 540 ggtctgcacc accgtgtgat cccgtacgcc tacttctcca ggtccgagca gtaccgcccg 600 atccgcttca acaccatcgt ggacgacgac acggccgcag ggcccgccga ggacaacgcc 660 cgggtccagg gtgacttcga cggcgacggc cgcgacgacg tggcggtgct ctacgactac 720 ggcaggaagg acgaccgcag tcgctcggcc ctgtggacgt tcaacagcaa cggcagcggt 780 ttcaacagtc ccaagcaggt gtgggacagc gggacgtcgg agagctggaa ctgggcctcc 840 agcaagttga cggtcggtga cttcaacggc gacggcaagg ccgacatcgg cgtcctctac 900 aacatgggcg cgaccgagga cggccgcaac cgcaccaagc tgttcgtgtt caccagcacc 960 ggcagcggat tcgccgcccc ggtcaaggtc tgggacagca acgacgaccc cgtgaagagc 1020 tggaactgga acgccagcaa gctcaccgtc ggcgacttca acggcgacgg caaggccgac 1080 atcggggtgc tgtacgacta cggcaaggac gacgaccaca accggacagg gctctggacg 1140 ttcaccagca ccggcagcgg gttcaacagc ccgaagcagg tgtgggacag caacaacgac 1200 cccgtgaaga gctggaactg ggaagccagc aagcccgtct ccggggactt caacggcgac 1260 ggcaaggccg acatcggcgt cctctacgac tacggcaaga ccgactccgg cagccgcacc 1320 ggactctgga cgttcaacgg caatggcaac gggttcaaca gcccgaagca ggtgtgggac 1380 agcaacaacg accccgtgaa gagctggaac tgggaagccg gcaagcccgt ttccggcgac 1440 ttcaacggcg acggcaagag cgacatcggc gtcctctacg acatgggtcg caccgaggac 1500 ggccgcaacc gcaccaagct gttcaccttc accggcacgg cgaccggttt caacagcccg 1560 gtcaaggtgt gggacagcaa cgacgacccc gtgaagagct ggaacgcgtc cgcgagcaag 1620 cccgtcgcag gtgacttcaa cggcgacggc aaggcggaca tcggcgtcct ctacgactac 1680 ggcaagaccg actccggcaa ccgcagcgga ctgtggacct tcaccagcaa cggcagcggc 1740 agcgacagcc ccaagcttgg ctgggacagc agcgcggacc ccgtcaagag ctggaactgg 1800 agcgcgagca agctcggctg a 1821 83 2466 DNA Streptomyces lavendulae 83 atgcgaacca tacgaatacg aagaacgaac ggcgtggcct tcgccgccgc tgccgccctg 60 atggccctcg tcgcctccgg caccgccacg gtccaggccg cgccctcgca cgccggaccc 120 tccggcacca ctccgatcac ctaccgtggc ctcaccctcg acataccctc cgggtggccg 180 gtcgtggacc tggagaaaga cccgcacacg tgtgtgcggt tcgaccgcca cacggtgtac 240 ttgggccacc ccggcaccga acagtcctgc ccctcccatc tggtcgcgga caagacggac 300 gccctgatat tggagccgat caccggagcg ggcggccagg acgcctccca cgcgctgcgc 360 atccctgccg gggccccgat gccgcacgag ctgccggtga cgtacgacca cgagacgaag 420 gtcgccgtcg aaggcgccgg agtcatggtc acgtcctcct acggcacgtc cagtacaacg 480 gtcgccgccg tcctcggctc ggcccgcacg gacgcgacag ccaagccgac ccccctgccc 540 ggcaaggcgg gcaggggcct cgcggctcca ccggttgccg ccgtcgcggc cgacaaggga 600 tacacagggc tgggcttcga gtcctgcacc gccccttcgt ccgccgcgat gaaggcatgg 660 aaggcctcgt cgccctacgg ggccgtcggc atctacatcg gcggtcgcaa gcggggctgt 720 gcgcaaccgc agctcaccgg cgactgggtg cgtcagcaga ccgccgacgg ctggcacctg 780 ctgcctctct tcgtggacct ccaggccggc gacatctctc cggccaccgc ggacgcgcag 840 ggccgcgagt ccgcggacgc cgccgtggcc aaggcggcgg acctgggcct gggccccggg 900 acggtcatct acagcgacat ggagcactac gacagccgct cgtaccgggc ccgggtcatc 960 gactacgtgt cggggtggac cagccgcctc cacgaacatg gctaccgctc cggtgtgtac 1020 gcgggtgaaa cgagcggcat cccggacctc gcctcggtgg ccgacgacac caactacgca 1080 tcacccgacg tgctgtggtc ggcgaactgg aacctcaagg ccgatgtgtc ggacgcgtcg 1140 atgggacttc cgggccccgg ctactggccc aatgggcggc gcatccacca gtaccgcggc 1200 caggtgaacg acacctacgg cggtgtcacc ctcgccatcg accgcgacta cgtcgatgtc 1260 gccgcggact cggccctgcc cgcacccggc ggagaggacg gttcctcgcg cgtcaagggc 1320 gacttcgacg gcgacggccg cgacgacgtg gccgtgctgt acgactacgg caaggagggc 1380 ggcgtcagcc ggtccgcgct gtggacgttc gcggggaccg gcagcggctt cggcgccccg 1440 aagaaggtgt gggacagcgg atcggacagc tggagttggt cggccgccaa gctgacggcc 1500 ggcgatttca acggagacgg caaggccgac atcgcggtcc tgtacgacat gggtcgcact 1560 gaggacggcc gcaaccgcac caagttgtac gagttcacca gcaccggcag cggattcaac 1620 agcccggtca aggtctggga cagcaacgac gaccccgtca agagctggaa ctgggcctcc 1680 agcaagctga ccgtcggcga cttcgacggc gacggcaagg ccgacatcgc ggttctgtac 1740 gactacggca gggacggcga ccgcagccgt acgggcctgt ggaccttcac cagcaccggt 1800 gccgccttca ccggccccaa gctggtgtgg gacagcaaca acgacccggt caagagctgg 1860 aactggaacg ccagcaagcc caccgtcggc gacttcaacg gcgacggcaa ggccgacatc 1920 ggcgtcctct acgacatggg tcgcaccgag gacggccgca accgcaccaa gctgttcacc 1980 ttcaccggca cggcgaccgg tttcaacagc ccggtcaagg tgtgggacag caacgacgac 2040 cccgtgaaga gctggaactg ggacgccgtc aaggtagtgg gaggcgactt caacggcgac 2100 ggcaagagcg acatcggggt gttgtacgac tacggcaagg acggcgaccg cagccgcacc 2160 ggactgtgga ccttcaccag caacggcagc gggttcaaca gcccgaagca ggtgtgggac 2220 agcagcaacg acccggtgaa gagctggaac tgggccgcga gcaagccggt cgcaggggac 2280 ttcaacggcg acggaaaaac ggatatcggc gtgctctacg actacggcag gaccgattcc 2340 ggcaatcgca ccggactgtg gaccttcacc agcgacggca ccggattcgg tacacccctc 2400 ctgggctggg acagcgtgac ggatgccgtg aagagctgga actggcgtgc cagcaaggtg 2460 agttga 2466 84 1575 DNA Streptomyces lavendulae 84 gtggatccct tgacgcgcaa gacccgcacc ccccgcaaga agggcagacg cgcgagcgcg 60 gcggcgatgt cggcctccgg catgctgctc gccttggtgg ccaccgccgc ccccgtcccc 120 gcccaggcgg catcactcgc cacctgggaa aagatggccc agtgcgagag cagcggggac 180 tggggataca accagccacc gtactacggc ggcctgcaat tcctggagag tacgtgggtg 240 gcgtaccacg gaacggacta tgcgccatac ccctatcagg ccaccaagga acagcagatc 300 cgggtcgcgc agcggctcct cgacaatgag ggcgcggctc cctggccgta ctgcggaaag 360 aaggtggggc tggctgacga cgacgcacgc cccttccccg acgcgccgga cgacgacgcc 420 tccgcccgga tcaacggtga cttcgacggc gacggatgcg acgacgtggc cgtgctctat 480 gactacggca aggagggcgg cgtcagccgg tccgggctgt ggacgttctc cgggagcggt 540 accggcctcg gcagcccgaa gaaggtgtgg gacagcggat cggccagctg gagttggtcg 600 gccgccaaac tggccgtcgg cgatttcaac ggcgacggca aggccgacat cgcggtcctg 660 tacgacatgg gccgcactga ggacggccgc aaccgcacca agttgtacga gttcaccagc 720 accggcagcg gattcaacag cccggtcaag gtctgggaca gcaacgacga ccccgtcaag 780 agctggaact ggaacgccgg caagctcacc gtcggcgact tcaacggtga cggcaagacc 840 gacatcggcg tcctctacga ctccggcaag accgactccg gcaaccgcac cggactgtgg 900 accttcacca gcaacggcac tggattcaac agcccgaaac aggtgtggga cagcaagagc 960 gacccggtga aaagctggaa ctgggccgcg agcaagccgg tcgcgggcga tttcaacggt 1020 gacggcaaga ccgatatcgg ggtgctttac gactacggca aagatggcga ccgcagccgc 1080 accggactgt ggaccttcac cagcacgggc agcggattca acagccccaa gcagacctgg 1140 gacagcgggt cggaaagctg gagatggtcg gcggccaagg tggtcggcgg cgacttcaac 1200 ggtgacggca aggccgacat cggggtgctg tacgacctcg gcaggaacgg cgaccgcaac 1260 cgcaccgaac tgttcacgtt cgcgggcaac ggcaccggcc tcaacacacc ggccaaggtg 1320 tgggacagcc aggacgacag cgcggtgaag agctggaact gggccgcgag caagccggtc 1380 gcaggtgact tcaacggcga cggaaagacg gatatcggcg tcctctacga ctacggccag 1440 accgactccg gcaaccgcac cgggctgtgg accttcacca gcgacggcag tggattcgcc 1500 ggccccaagc tcacctggga cagccggacc gaccccgtca agagctggaa ctggaacatg 1560 agcaagaccg gctga 1575 85 2175 DNA Streptomyces lavendulae 85 atgaaatacc gaccgggaac actgctcact tccataacag tcttgtgtgc cctgctcgtt 60 ccggtgcgtt cggcggctca ggcggccagg cccgagcagg gacgttccgt ggtggccgcg 120 gccgccgtac tggagcaaag tccgccgacg ctgctcgccg agccggaaat gcgcgtcgtc 180 tcctggaaca tctgcggtga ggcgggcggg gtgcgcgggg agggcggcta ctgcccctac 240 cgcaacgatc cccaggcgaa agtcgaccag atcgcgcagg tggtcgcgga gcgcagtgcc 300 aatgtcgtca tgctccagga agtgtgcggc gaggcgcccg gcagccatat ggagcggctg 360 cgcgcggccc tgggcagcgg atggtcgatc gcgcacgccc cgggggcccg cccggacgac 420 ggaaccacga actgccgggg cgggctcagc ggcatattgg gcgtggggat cgcggtgaag 480 gggcgcgtca ccgacaccac cgcgacgaac accgtgcccg ggggcggcgg tgacaagcag 540 accctgccca tcctctgtgt acgtgtcgag ggctggtcgt ccaggatctg caccacccac 600 atcctgtccg accctgccga tccgcgcagg ccggggcaga tccagaacgt caagaacgag 660 atctggccgg accgctatca gctggtgctc ggcggcgact tcaacatgtt ccccgactcc 720 gccgggctca agccgatctc ggacgaattc gacgagtgcg accgccgctc ctacggcgcc 780 ggtgacatgg tcaacgaggt cacccatcac tcctgggaga aaaagggcgg acacatatgg 840 cgcaagcgtg accacatctt cgcctcgtgg ggagagtccg ggagccagtt cacatcctgc 900 gaggtcgacc ggacccggat ggacaccacc gagaacgcgc ccgaaagcgg tccgcccaac 960 gggtattcgg accatgcgcc gatcatcggc tacctcaagc cgccgcggca cctgagcacg 1020 tccggggact tcgacggcga cggcaaggcc gacctcgcgg tcctctacgg gcaggggaag 1080 accccggacg gccacaaccg gtccagcctg tggatctcag gcggttccgg taccggagcg 1140 gagaccggat tcgccgcgcc gcgcgaggtc tgggacagcg gtgccgacag ctggaactgg 1200 tccgcgagcg cgctgacctc cggggacttc gacggcgacg gcaagaccga catcggcgtc 1260 ctctacaact acggcaggga cggcgaccgc aaccgcaccg cgctgtggac cttcaagggg 1320 acatcgaacg gcttcgaggc gccccgcaag gtgtgggaca gccacgacga cacggccgtt 1380 cccagctgga actggtccac gagcaagctc gtcgcgggcg atttcaacgg cgacggcaaa 1440 gcggacatcg gcgtcctgta cgactacggc aggaccgcct ccggcaaccg caccggactg 1500 tggaccttca ccagcaccgg caccggattc ggcaagcccc acctggcgtg ggacagctcc 1560 accgacccgg tgaagagctg gaactgggcc gcgagcaagc cggtcgcagg tgacttcaac 1620 ggcgatggca agaccgacat cggcgtcctc tacgactacg gcaaccacac cgccctatgg 1680 accttcacca gcaacggcac cggattcgcc ggccccaagc aggcctggga cagcggaccg 1740 gagaactgga actggtccgc cgccaagccg gtcgccgggg acttcgacgg cgacggcagg 1800 accgacatcg cggtcctgta cgactacggc aggaccgcct ccggcaaccg caccggactg 1860 tggaccttca ccggcaccgg caccggattc ggcaagcccc acctggcgtg ggacagctcc 1920 accgacccgg tgaagagctg gaactgggcc gcgagcgagc cggtcgctgg tgacttcaac 1980 ggggacggca gggccgacct cgcggtgatg tacgactacg ggaacgcgac caacggccgc 2040 aaccgcaccg cgctgtggtc cttcaccagc cgcggcacgg acttcgccgc cccgcgggcg 2100 aactgggaca gcagcaacgc cgctgaccag ctgaaatcgg gcgagctgag ggcggctccg 2160 ctcagcgggt cctag 2175 86 27 DNA Artificial Sequence A primer 86 tcagaattcg gatccgaggg ccggagt 27 87 18 DNA Streptomyces lavendulae 87 acctactgcc tcgatgcc 18 88 15 DNA Streptomyces lavendulae 88 ctgatccttc aagcg 15 89 20 DNA Amycolatopsis mediterranei 89 gcgtccgtgc tgcgcgcgca 20 90 20 DNA Amycolatopsis mediterranei 90 tgcgcgcgca gcacggacgc 20 91 6 DNA Streptomyces lavendulae 91 gaaagg 6 92 80 PRT Artificial Sequence A conserved motif 92 Gly Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Glu Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa Gly Glu Gly Gly Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly 65 70 75 80 93 6 DNA Streptomyces lavendulae 93 ggaacg 6 94 30 DNA Artificial Sequence A primer 94 gggaattcca tatgatgcag tccatgtcac 30 95 30 DNA Artificial Sequence A primer 95 gggaattcaa gctttcattc cgccggggtc 30 96 18331 DNA Streptomyces lavendulae 96 ggatcccgat cgtctcggac atgaccggcg accttctcgg cgcgcgggag gcccaggacc 60 ccgcctactg ggtgtcccac atccgccgcg cggtgcgctt ccacgaccag atccgccgtc 120 tgcagcgcta cggggccggg gccttcgtcg aggtcggccc ggacacggtg ctcagctcgg 180 ccggccaggc gtgcctgacg gaccaggcgg gcaggagcgc gcccgtcctg gtgtccctcg 240 cgcacgccga gcgcgcggag gtgcccgcgc tcctgaccgc tctggccacc ctgcacaccc 300 gtggcgtggc cgtggactgg cgggcgtggt tcggcgacgg gccgcgcgcg gccggcctgc 360 ccacatacgc gttccagaag cagcactact ggccgtcggg ccccaccggt tggcggtccg 420 ggcccgcccc cgtacccctg ccccaggccg gaacggagga cgccgaaagg cccggtcgcg 480 ccgcggagtg gcgggcgctg ccgcccggtg agcggtacga cgcgctgctg cggatggtgc 540 gcggcgaagc cgccgccgtg atggggcacg ccgggccgga ggcggtggag ccggagcgcg 600 gcttcctcga ccacggcttc gactcggtga tggccgtgaa gctgcgcgac cgtctcgtgg 660 ccgggacggg gcgggagctg ccgacgaccc tgctgttcga ccaccccacg cccgcggccg 720 tcgccgacta cctgctggcg gggacgggcg aggccgagac ggcgccgtcc gtgtccctgt 780 cggaccagct cgaccgcctg gaggccgacc tcgcgcggct gccggccgac gaccggcagc 840 gcgcccgcgt cgccgagcgg ctcaagggcc tgctcgcggt ccacgcgccg gaccggggcg 900 ccgggagcga ggacgcgccg gaccaggacg cgctggacac ggcgaccgac gacgagatgt 960 tcgagctgat cgagaaggaa ctccgccgtg gatgagacca acgagaccaa actccgcgag 1020 tacctgcggc tggtcacggc cgatctgcgg cgaacccgca ggcagttgga ggaggccgag 1080 gacgcggccc gcgagcccgt cgcgatcgtg ggcatggcgt gccgcttccc cggggacgtg 1140 gcatcgccgg acgacctgtg gcagctggtc gccgagggcc gggacgccgt caccgagttc 1200 cccgccgacc ggggctggga cgtcgacgcc gtctacgacc ccgagccggg caccccgggc 1260 aggacgtacg cgcgccacgg cggcttcctc aaggacgccg ccggattcga cgccgccttc 1320 ttcggcatca cgccgcgcga ggcgctcgcc atggacccgc agcagcgcat gatcatggag 1380 gtctcctggg aggcgttcga gcaggcgggc ctcgacgcga ccaccctgcg gggcgaggac 1440 gtcggcgtct tcgtcggctc caacagcaac gactacctga tcaacgtgct cgacgcgcgg 1500 gacgtcgccg agggcttcat cgggaccggc aactccgcca gcatcctctc cggccgcgtc 1560 gcctacacct tcggcttcga gggcccggcc gtgtccgtcg acaccgcctg ctcctcctcg 1620 ctggtcgcgc tgcacctggc cgcgcagtcc ctgcggcagg gggagtgctc cctggcgctg 1680 gcgggcggcg cgacggtgat ggccacgccg accgccttca tcgagttcag ccgccagcgg 1740 ggcctggccc ccgacggccg ctgcaagtcc ttctcggcga ccgccgacgg caccacctgg 1800 tccgagggcg cggccgtgct gctgctggcc cggctctcgg acgcccgccg cctgggctac 1860 cccgtgcacg cggtcatccg gggcagcgcc gtcaaccagg acggcgcgag cgcgggcctg 1920 accgcgccca acggaccggc gcaacagcgg gtgatccggc aggcactggc caacgcacgg 1980 ctgacggccg acagcgtcga cgcggtcgag gcacacggca ccggcacccc gctgggcgac 2040 ccgatcgagg cccaggccct cctcgccacc tacgggcggg cccgcggcga gggcaggccg 2100 ctgtggctgg gctcgctgaa gtcgaacctg ggccacaccc agtccgcggc cggcgcgggc 2160 ggcgtcatca agatggtgat ggccatgcgg cacgggacgc tgccccgcac gctgcacctc 2220 acggagccca ccccgcgcgt cgactggtcc gccggtgacg tacggctgct gaccgaggcc 2280 caggactggc cggacaccgg acagccgcgc cgtgcggccg tctcgtcctt cggcgtcagc 2340 ggcaccaacg cccatgtgat cctggagggc ccgcccgccg aggaggcacc ggacgcgccg 2400 ctgccggacg tctcctcgca gccgcggggc ccgctgccgt gggtcgtctc cggccgcagc 2460 gaggcggccg tccgagcgca ggccgagcgc ctggcggccc acctgaccgc gcgcccgcac 2520 ctggcaccgg ccgacgtggc caccgcgctg gccaccacgc gggcggcctt cgaccaccgg 2580 gccgccgtcg tcggccggga ccgtgaggaa ctgctcgccg gcctcgcggc cctggccacc 2640 ggaacccgcg cgcccggcct ggtcaccggc cggaccccgc cgtccggcgg caaggccgcc 2700 ttcctcttca ccggacaggg cagccagcag cccggcatgg gccgcgaact ggcggctcac 2760 agcaccgtgt tcgccgacgc cctggacgag gtctgcgccc agctcgaccg gcacctcgac 2820 cggccgctgc gcgaggtgct gttcgccgcg gacggcacgc ccgaggccgc cctgctcgac 2880 acgacggcct acacccagcc cgcgctgttc gccgtcgagg tcgcgctgct gcggctgctg 2940 gaggactggg gcttgcggcc cggcatggtc gcgggccact cggtcggcga actgaccgcc 3000 gcctacgccg ccggggtctg gtcgctcgcc gacgcctgcg ccctggtcgc cgcccgcggc 3060 cggctgaccc aggcactgcc cgcgggcggc gccatggtcg ccgtgcaggc gaccgaggac 3120 gaggtgcgcg cccaactcgc cgacggccgc cccggcgtgg acatcgccgc cgtcaacgga 3180 ccggaagcgg tggtgctgtc cggcgacgag gccgccgtca cggacctggc gcgcgagtgg 3240 gccgcccgcg gccgggagac caggaggctg cgggtcagcc acgccttcca ctccgcccac 3300 ctggacgcca tgaccgaggc gttcgccgag gtcgcacgag gggtgtccta cagcgcgccg 3360 tccctcccgg tggtctccac gctcaccggg gcccccgtca ccgacgagct ccgcaggccg 3420 gaacactggg tgcggcacgt ccgggagacg gtgcgcttcc acgacgcggt ccgcgccctg 3480 cgcgaccgcg gggccaccgc gttcctggag gtcgggcccg gcggcgtgct gacggccgcg 3540 gcacgccgat gcctgcccga cgccgccccc gagacgttcg tccccgtgct gcggcgccgc 3600 aggcccgaac ccgagtccgt gctgacggcc gtcgcgcagg cccacacgat cggcctctcg 3660 ccggcgtggg accgcctgct gcccaaggcc cggacgcgcg tggacctgcc cacgtacgcc 3720 ttccagcgcg gccactactg gctggcgggc atggccggag cgggcaccgc gcggccggtg 3780 cggccggaag tgcaggagcc caccgccccc tccggtacgc cgccgctgtc gcgacggctg 3840 gccgacgcgt cggaggagga gcgcggccac ctgctgctga cgctggtacg cgagcagtcg 3900 gccaccgtga tgggcggcgt cgaccccgcg caggtcgaac ccgaccgccc cttcctggag 3960 ctcggcttcg actccctgat gggcgtcgag ctgcgcaccg cgctcgccgc cgactgcgca 4020 ctgcccctgc cgcccggcct gatcttcgac caccccacgc ccgccgccct ggccgccttc 4080 ctcggcgagc agctcgcggc ggcggcctcc ggcaccccca cggcggcggc accctcgccg 4140 tactccctgg aggcgctgta ccgcaacgcc aacaccctcg accggcccga ggacgcgctc 4200 gccctcacca aggccgcctc ccggctgcgc ccggtcttcg ccagcgtggc cgaggcgggg 4260 caggacccgg tcacggtgga gctggcacag gccaccggcc ttccgggcct gatctgctgc 4320 ccggcacccg tgccgctgta cggggcacag cagtacagcc ggctcgcagc cgccttccgc 4380 ggcacgcgcg gagtctcggc cctgctcgcc cccggcttct ccccgggcga actgctgccc 4440 gccgacttcg aggtgatgca ggacttcctc gccgaggggg tccggcggca gaccgacggc 4500 gcgcccttcg tcctcctggg ccactcctcc gggggctggt tcgcctacag cctggcggcc 4560 cacctggcgc gcaccgggcc gcgcccggag gccgtcgtgc tgctggacac ctatcagctg 4620 cacgacccgg cgctgcaccg catgcagcgc gaactcgccc agggcgtcct ggaccgcgag 4680 gaggacttcg gggcgatgac ggacgtacgg ctgagtgcca tgggcaaata cttcgacttc 4740 ttcaccgact gggtggccga ggacgccggt gtcccgacgc tgctgctgcg ggcctccgag 4800 cctctgggcg aggtcgtcga gggccaggag tggcgctcca cctggccgtt cgacagcacg 4860 gtcctcgaca cggaaggcga ccacttcgcc atggtcaacg accacgcgcc gcggacggcc 4920 caggccgtga acggctggct gtcgggcctc accggcggaa ggggctgagc gccggtggag 4980 acacgcaacg ccgaacggcc gtggatacgc agcttccacc ccgctcccca ggcccctgtg 5040 cggctgctgt gcctgccgca cgccgggggc tccgcgagcg cctacttcgc gctgtcgagg 5100 gaactggcgc cccgggtgga ggtgctcgcc gtgcagtacc ccgggcggca ggaccggcgc 5160 gacgagccgc tgctggactc gatcgaggcc ctgcgcgacg gggtcgccga ggccctgacg 5220 ccctggctgg accggccggt cgccctcttc ggccacagca tgggcgccgt ggtggcctac 5280 gagctggcgc ggctgctgtg ccaggacgcg ggcgtgccgc tcacccacct cttcgtctcc 5340 ggacgccggg gatccgaccg aagtctccgt ccttgccgcc gtgttccgga attcaccgtg 5400 acaccgccgc gcggctcttc ttccgaagtc ctccagatcc ggcacgagtt tgtatccgaa 5460 cggggttctg cgtgcgaaat actctcttcg aattgggtga catacccccg atcggcaccg 5520 tacccgagca gatgtacgcc tcggtgatcc gacgggagcg ctacggacag ccccaccagg 5580 cgttccgcag cgaggtcgtg gacgtgccga aggtggggcc cggtcaggcg ctggtcctcg 5640 tgatggccgc gggcatcaac tacaacaacg tctgggcctc cctggggcag ccggtcgacg 5700 tgatctccgc gcggcagaag cagggccaca gcgaggactt ccacatcggc gggtccgagg 5760 gctccggcgt ggtgtgggcg gtgggggagg gcgtcaccca ggtcgcggtg ggcgacgaag 5820 tgatcctctc cggctgccag tggacggaga cggccgccga catccggctc ggcgccgacc 5880 ccatgacctc cggctcgcag tcggtgtggg gatacgaggg caactacggc tccttcgccc 5940 agttcgccct cgtcgacgac tatcagtgcc accccaagcc gcccggcctg acctgggagg 6000 aagccgcctg cttcctgctc accggggcca ccgcctaccg ccagctgtgc ggctggcagc 6060 cgcacgacgt gcggccgggc gacccggtcc tcatctgggg cggggccggc gggctcggct 6120 ccatggccat ccagatcacc cgggcgcggg gcggcatccc cgtcgccgtg gtctccgacg 6180 aggagcgggc ccgctactgc cgggagctcg gcgcccaggg caccatcaac cgcctggact 6240 tcgaccactg gggacggctg cccgacatcg gcgaccacga ggcgatgggc cgctggaccg 6300 agggtgtacg ggccttcggc cggcgcttct gggaggtgct gggcgagcgc aggtccccgc 6360 gcatcgtcct ggagcacagc ggccaggcca ccatccccac ctcgatgtac ctgtgcgaca 6420 acgcgggcat ggtcgtcatc tgcggcggca ccaccggcta caacgccgac atcgacctgc 6480 gcttcctgtg gatgcgtcag aagcgcttgc agggctcgca cttcgccaac ctgcggcagt 6540 gccgcgacgt catccacatg gtcgcgaacg gccagctcga cccgtgcctg tcgtggaccg 6600 gcggcttcga cgacatcggc aaggcacacc agctgatgca cgacaaccag cacccccagg 6660 gcaaccaggc cgtcctggtc aacgcgccgc ggaccggcct gaccaccttc gcctgaacca 6720 ccgccccggt gttccgacgt cttcccccca cacttaccga ccaaggagag atcaccatgg 6780 acaagctcga catcctctgg agcgagcgcg agatccgtgc cgtgctgcag cgctactgcc 6840 gcgggctcga ccgcctcgac gaggaactgg tcaagtccgc ctaccacgag gacgcgcacg 6900 acgaccgcgg cgtcatccgc ggcaacgcac acgacttcgt caagcagatc gtcccgctcc 6960 tgcgcgacgc ctacaccggc accctgcaca ccctgcacgg cagcacgatc gagatcgacg 7020 gggatgccgc gggcgtggag tcctactgca ccgcctacca ctaccgcgag agcgacggca 7080 tcaagcgggt ggagcagttc gccgggcgct acgtcgaccg cttcgagcgg cgcgacggcg 7140 tctggaagat cgcccgccgg ctcgtgctga acgacttcag cctcgcccag gaggtgccgc 7200 tcgaccccgc cgaggcccag gccggcttca acccctccca ccgcgacctc accgacgcca 7260 gctaccaggt gctgccgctg cgcggcccgg acgcccccac cctctgagcc gtccggccgc 7320 cccaactcgc cccacctcac caggagtcac caccgtgtcc gacaccgagc agcacgcgcc 7380 cacgctgccg cggcagcgca cctgcccctt ctcgccgccg cccgagctcg aggagctgcg 7440 gcgcaccgat cccatcagca ggatgcggtt cgccgacgac tccccgggat ggctgctgac 7500 ccgccacgcc gacgtccgcg ccgcgctggc cgaccccggc gtcagctcgc accccggcaa 7560 ggcaccccag ccctggcgca acctcgcccc cgagatgcgc gccgagcact acctgccggg 7620 cttcctgatc ttcatggacc cgccggacca cacccgctac cgccgcctgc tcaccaagtg 7680 gttcaccatg cgggccatcc gcaagctcga acccaggatc gagcagatcg tcaccgagac 7740 cctcgacgcc atggaggccc agggcggcac cgtcgacctg gtgcagtcct tcgcgctgcc 7800 gatcccgctg ctggtcatct gcgagctgat gggcatccgc tacgaggagc gcgaggagtt 7860 catggacatg gtcctgcgac tccaggccct ggacgccacg cccgaggaac tcggggccct 7920 cggcgccagg atgaacgagt tcatgatgaa gctcgccgcc gccaagcgcg cgaaccccgg 7980 cgacgacctg ctcagccacc tcgcccacga ccccgacgcc gacccggcgc tcacggatct 8040 ggagatcgcc ggcatcggcg tgctgatgct catcgcgggg cacgagacct cggccaacat 8100 gctgggcgtc ggcacctaca ccctgctgga gaacgccgac cagtgggccc tgctccgtga 8160 cgacatcagc ctgatcgacc gggccgtcga ggagctgctg cgccaccaga ccatcgtcca 8220 gcagggcctg ccgcgcggcg tcacccggga catggagatc gccgggcacc aggtgaagac 8280 cggggagtcc ctgctggcct cgctgcccgc cgccaaccgc gaccccgccg tcttccccga 8340 ccccgaccgc ctcgacatca cgcgcgagca caacccgcac ctcgccttcg gccacggcat 8400 ccacctctgc ctgggcatgg agctcgcccg ggtggagatg cgccaggcgt ggcgcggcct 8460 cgtcacgcgc ttccccggcc tgcgcatggc cgccgcgccc gaggacatcc gctggcgcga 8520 cgaccagatc gtctacggcg tgtacaacct cccggtgacc tgggacgagg ccaagtgacc 8580 ggccccgagg ccgcggtgcg cgggtgcccc ttcggcgccg gcgaggcgcc cgcgtacccc 8640 ttccacgccc ccgaccggct ggagcccgac ccgtactggg agccgctgcg ccgcgagcgg 8700 ccgctgcaac gcgtcacgct gccgtacggc ggcgaggcgt ggctcgccac ccgctatcag 8760 gacgtgcgcg cggtcttcgc cgaccgcagg ttctcccggc agctcgccgt cgcgcccggc 8820 gctccgcgct tcctcccgca ccagccgccg ccggacgccg tcctgagcgt cgagggcccc 8880 gaccacgcgc ggctgcgccg gctggtcggg aaggtcttca cgccgcgccg cgtggaggac 8940 atgcgtccgc tcatccagcg caccgccgac ggactcctcg acgcgatgga ggagatgggg 9000 ccgcccgcgg acctggtcga ggacttctcc ctgcccttcg ccgtgtccat gatctgcgag 9060 ctgctcggcg tgccgcccga ggaccgcaag cggttctgcg tctggtcgga cgcgctgctg 9120 acgaccaccg cgcacacccc cgcccaggtg cgcgactaca tgatgcagat gcacgactac 9180 ctcggcgggc tcgtcgcgca gcgccgggtg cggcccaccg cggacctgat cggctccctc 9240 gtgaccgcgc gcgacgagga ggacaagctc accgagggcg agctggtgcg gctggccgag 9300 gccatcctca tcgccggcta cgagacctcg gcgagccaga tccccaactt cctctacgtc 9360 ctcttccgcc acccgcagct gctggagcgg atcaggaacg accacgacct catccccgac 9420 gccgtcgagg aactgctgcg cttcgtgccc atcggcaccg tggacggctt tccccgtacg 9480 gccaccgagg acgtcgagct cgggggagtc ctggtcaggg ccggggagac ggtcgtgccg 9540 tcgatgggcg ccgccaaccg cgaccccgag ctgttcacgg accccgacga gctggacctc 9600 gcgcggcggc cgaatccgca cctgggcttc ggcgcgggac cgcaccactg cctgggcgcc 9660 caactggccc gggtggagct ccagatcacg ctcacgacgc tgttccgcag atacccccgc 9720 ctgcggctgg ccgtgccgga ggagagcctc tcgtggaagg aggggctgat ggtccgcggc 9780 atgcacacca tgccggtcac ctggtgagga caccggcgtc ctcctgacct tcccggcgtt 9840 ctcacgccgt cccggcagcc ttccttccga cacgagcgca cagagggtga agcgaccgca 9900 atgagcacca tcgacgaatg ggaacacagc acgaaggagg cgggcatgga ccccgcggcc 9960 ctcagacgcc tgaccgatgt ggtgcgggcg aggggcggcg cggcgcagct gtgcgtcatg 10020 cggcggggca ccgtggtcct ggaccgctcg ttcggctgct cctccgactc cctcttcctc 10080 gtctacgcgg ccaccaagcc cgtcgccgcc ctcgccgtgc acgcgctcgc cgagcggggc 10140 ctgatcgggc tggaccggcc ggtggccgaa tactggccgc agttcgcccg gcacggcaag 10200 ggtgacgtga ccgtccgtca tgtcctccag caccgggccg gggtgccggt cggccggggc 10260 atcgtgcgca cgatgcgcac cgccggcgac tgggagcgct ccgtgcgcga ccttgagcag 10320 tcccggccca agtggcccgg cggcgaggtc gccgcctacc acttcatgag tttcggattc 10380 attctcggcg aactggtgca gcgcgtcacc gggcggtcgt tccgagattt cgtgacttcc 10440 gagctcttcg ccccacttgg gctgaatgat ttgcacatgg gattgcccgg cagtgcctgg 10500 ccccggcatg tgcccgcgcg ggccgcccac ccctccgaat ggcccaatca gtggatgagc 10560 aaccgccgcg gctaccgcca ggccgtcatt ccgtccgccg gtctttccgg aaccgccgca 10620 caaatggccc gcttttacca gatgcttatg gagggcggct cgctcgacgg catccgcgtg 10680 ctgcggcccg aaactgtgga ggaagccaga aaaccgtcca atgacggcgg aatcgacgct 10740 tccctcaagc gtccggtccg ctggtcccac ggattcatgc tcggtggtcc gggcccggac 10800 ccgcgggggc tgtccaatgt gctgggccgc acgagcgacc cgagcgcctt cgggcacgcg 10860 ggcaccacgt ccagcgtcgt gtgggccgac cccacgcgcg agctggtcct cgcctacctc 10920 tccaacatcc agcccgagtt cggagcgggt atcgagcggc tccgcgaggt cagtgacctc 10980 gcgctcggtg cctgcgaggc aggctgaccc gagccgtgcc gccacggccc ggcgcccgcc 11040 cgatccgatc gggtccggtg ggggccggcc gggtccgggc ggggacgcac ttcccccggc 11100 gtccccgccc gggccccggt gcgaaccggg cgcaaaggcg gccgatcgcc cggcgcggcc 11160 ggatgccccc gaacggtgtg aaacgttctt atcagcctct gaccagcacc gagtgatcta 11220 ctgcacagcc cgaggccgcg attccggcag tatcttgatc ttgacggggc accaatgcga 11280 gcgggctatt cgccgcggtt ttccctgacg tcggatgcag atgacaccgg aggagggcca 11340 gtgctgaatc tgcccaaagg aatggagcgc gcgcatccgc attctccgcc acaggtggga 11400 atactcggac ccttggaagt ccgctcggcc ggaggtgccg gaacgggagc cgcggtaagc 11460 ggtattcgcg tacgcacatt gcttgccgcg ttgactgccc gcctggggca ggcgatgtcg 11520 accgagcgca tcctcaaaga ggtctgggcc gacaacccgc ccgcgaccga tcgcaaggcg 11580 gtggccgtcg ccgtcctgcg gctgcggcgg gtcctcggcg acaacgaagg acggtggctg 11640 ctcacccgcc cctccggtta cgtcctggac atccccccgg accacctcga cgccgtacgc 11700 gcggagaccc tggtgcggga aggccgggcc gccctggccg ccggcgaccc acgcgtcgcg 11760 gcccgccacc tcacgcgcgc cctcgaccag tggcggggcg agccctacgc ggacgccaac 11820 gccatctcga ccgtgtccca gcgcatcacg gagctggaga acctcaggtc cgaggccgtc 11880 caggcgcaca tcgacgccag gctcgaactg ggtcaccacc aggaactggt cggcgaactc 11940 cgctcgctga ccgccgcgaa ccccctgcac gagccgcact ggctgcagct gatgctcgcc 12000 ctctaccgct ccggcaagca ggccgaggct ctcgccgcct atatgcagct gcggcaggcg 12060 ctggccgaga acctgggcat cgacccgggt cgtcagctcc aggaactgca cctgcggatc 12120 ctgcgcgccg acgcgggcct gctgacgggg tccgggccgg cggcaccggc cgagccactg 12180 ctcgtacggc agtcctgagg gctcacggcc acccgaagaa cgcgcggtag cacggaacct 12240 gctgctccag catatggatg ccgtggtgca cacggcgccc ggcggtggcg gccgcgctca 12300 gcagcgccgt ctcgtgcggc ttcatgacga cgtcgaccac cacggcatcc ggtcgcaccc 12360 tcgcggggtc gaagggcagc gggtcctcgg aacgcatgcc cagaggcgtc gcgttgacgg 12420 cgaaatcggc cgcctccaga tcgccgggcc ccagcgcccg gatcccgtcc ggccggcggg 12480 acccgagccg cagcagcagc gcgtcgagct gggcgcggtc ggtgtcgtgc acggacaccc 12540 gcgcggcgtc ggccatcagc agcgccgtgg cgatcgcgct gcccgcccct ccggcgccga 12600 ccagtgccac atgcctgtcg cgcaccgtgt gcccggccgc ctgaagaccc tggacgaacc 12660 cgagcccgtc gaagttctcg gcgtaccagc ggccgtcggg ttcgcgccgc atcgcgttgg 12720 ccgtcccgat gagggcggcc gccggcccga gcccgtccgc gagcccgcac agggccgcct 12780 tgtgcggcac ggtgaccagc agaccgtcca gattgccgat ccgcttgagc ccctcgacca 12840 cctcggcgag atcccgcgcc cggacgtgca ccggcaccac cacggcgtcc agaccgcttt 12900 cgctcagcag ggggttgagc agaccgggcg ccttgacctg ggcgacggga tcacccagca 12960 ccgcgtacag ccgcgtggcg cccgagacac cggccgccgg cccgaggaat tccatcagcc 13020 gatcctctct gtacccccga cggatgttgc cctacggtgc tggagatgct ccacagcttt 13080 gccgtgaccg ccggtcggca caaccctgcg tgcccctgac gcgccaggcc ctccaggtag 13140 ttgctcccgg cggatcccga cagctcccga ccggtcccga cggagggaag aagccatcag 13200 atacctggga atcgacgtcg gaggcacgaa ggtcgccctg cgggtgacgg gggacaccga 13260 cggtgcgggc ggcggcgacg tgacgttccg ctggcccgcc gccggcgacg tcaccgcgga 13320 tctggacctg ctcgccgcgc gggtccgcgg tcttctggga caccgcgagg accccctcgc 13380 cggggtcggc gtggccatgc ccgcgatctg cgacgcggcc gggacggtcc gcacgtggcc 13440 gggacggccg agctgggcgg gcctgaacct gacggccgcc ttcgggcagt tgctgcccgg 13500 caccccggtc gcctgcgccg acgacggtga cctggccgcg ctggcggagt cccgcgccgc 13560 cggctgccgg catctgctgt acgtgggggt cggcacgggc atcggcggcg gcatcgtcca 13620 tgagggccgc gcctggccgg gccccggacg cggctcgtgc gaggtcggcc atgtcgtcgt 13680 cgaccgctcg ggcccacgct gcgactgcgg gcgcgccggc tgcgtccagg cggtcgcgtc 13740 gggaccggcg accctccggc gggccgccga acggcgcggc cgggagaccg gcttcgacga 13800 actggcctcc ggggcgcgct tgcacgcccc gtgggcggaa gcggccgtcg acgagagcgc 13860 cgcggccctg gccaccgccg tgaccggcat ctgcgagctg gcccaccccg aactcgtcct 13920 cgtcggcggc gggttcgcgg cgggcgtgcc gggatacgtg gcctcggtgg cggcgcacgt 13980 cgagcggctg acccgcccgg gaacggatcc cgtgcgggtg cgcccggcgg tgctcggcgg 14040 gcggtcctcc ctgcacggcg cactgctgct cgcgcgggag gcacacgggc ggggaaaccg 14100 gccgccggag agtgaccgtg tttcttccga tgtttcttcc gatgtttctt tcgggggagt 14160 gacagacagg gccgttggcc ggtccgactg agcacaatca caggtgattt cgcccaggtt 14220 caccacgcct cgtgtgctcg gggtcggcag aaggagtcag agtcatgctc gacaggcgga 14280 gcgtcattcg cgtcggcgcc ggggtggcgg cggccgccgc cgtggccggt acggccgcca 14340 ccggtgcggc ggccgtgggg ctgccgggtg tacggggacg cgcggcgtcg cgcggggtcg 14400 actgggcctc cttacgccgt catctgtcgg gcgagctcgt cctgccggcg gacaccggat 14460 acgagcgggc caggaagctc tacagcggcc agttcgacgg catccgcccg caggccgtcg 14520 cctactgccg gaccgaggag gacgtgcgga cgaccctcgc gttcgcccag gaccacgcgc 14580 tgcccctcac cccgcgcagt ggcgggcaca gcttcggcgg ctactccacg accgacggaa 14640 tcgtcctgga cgtctccggc ttccacgcgg tgagcctcac ccggaacacc gtcgtcatgg 14700 gcgcgggcac ccagcaggtg gacgccctca ccgccctgtc gccgcgcggt gtcgccgtgg 14760 cgagcggcaa ctgcgcgggc gtctgtcccg gcggcttcgt ccagggcggc ggactgggct 14820 ggcagagccg caagttcggc atggcgtgcg accggctcgt ctccgcccgg gtcgtgctcg 14880 ccgacggccg cgccgtgacc gcctccgcca ccgaacaccc cgaccttttc tgggcgatgc 14940 gcggcggagg cggcggcaac ttcggcgtcg tcaccggctt cgagctgcgc cccaccgacg 15000 tcccctccgt cgtcagctac aacctcacct ggccgtggga gtcggcgcgg cgcgtcatcg 15060 aggcgtggca gcactggatc atcgacggcc cccgcgacct cggtgccgcg atggccgtgc 15120 agtggcccga cgccgggacc ggcacgccgg tcgtggtcgt caccggcgcc tggctgggcg 15180 cggccgacgc gctcaccccc gtgctggact ccctggtggc ctccgtgggc agcgcgcccg 15240 ccacccgctc ggccaaggcg ctctcccagc acgacgcgat gatggcgcag tacggctgcg 15300 ccgacctcac gcccgagcag tgccacacgg tcggctactc gcccgaggcc gcgctgcccc 15360 ggcagaactt ctccatggac cgcaaccggc tcttctcccg ggccatcggg caaggaggcg 15420 tcgagcggat cctggaggcg ttcgccgccg acccgcgcgc cggacagttc cgcttcctga 15480 gcttcttcgc cctcggcggc gccgccaacc gccccgaccg caccaccacc gcctacgttc 15540 accgcgacac cgagttctac ctcggtttct cgatcgggct gaacgacccg gagtacacgg 15600 cggaggacga gaggctcggc cgcgcctggg ccgcgcgagg actgcgcacg ctcgatcccc 15660 actccaacgg cgagagctac cagaacttca tcgacccgga gctcgacgac tggaagtcgg 15720 cctactacgc cgagaactac gtgcgcctgg ccgccgtcaa ggcggcctac gacccgcacc 15780 ggctcttctc cttcgcgcag gccgtctgac ctctcccgaa agacccctgc cggcctgctc 15840 ccctccgcgg ctcctgtggg cactggtgcg cccgcgcact tctgtgtgat tgagtgaagt 15900 ccgggcgtgc agagctcagt tgccgtggag ggggcgccag ttgcgagcat cagcggtgga 15960 gagggtggag ctgatccgct ggccggtgga gtccgagcgg cgggagcgct gccgcgaccg 16020 gggcgtcatg cggatcctgg tgctggaggc gggggccgag gcacccttgt gcgtggaccc 16080 caaggaggac tgggtccgcg ctcccgtcag caccgacgac ctgcgggccc gcgtcgaggc 16140 cctgcgcctt cggggagccg ccgccgagtc ccggcccgag gtcgacccga acggagtgct 16200 gcgtttccgg tggcgctccg ccctgctctc gcccaccgag gcccggctcg tcgcccggct 16260 cgccgagtcc tatgccgagg tcgtcgcccg cgacgacctg ctccgcccgc ccccgggccg 16320 taccgtgccg agccgtaacg cgctcgacct ccacatcatg cggatccgac ggcgcctcgc 16380 cgcgctgggc ctgagggtgc gcaccgtccg ggggcgtggc tacgtcctgg agagcgcgga 16440 aggagtctga ccgacgggcg tggccgcgca ccgcaccgac cgcccctacg agcgaggagc 16500 ccgaagtgca gcagcctcat cacagccgcg tcgacgtgga actgggcgag aggtcctacc 16560 ccgtccacgt cggaccgggg gtccgccacc tcctgcccgg catcgtcgcc tccctcggcg 16620 cgcaccgcgc cgccgtcgtg accgcacggc cccccgacct ggtgcccgat cccggcgtgc 16680 ccgcgctgat cgtgcgggca cgtgacggcg agcggcacaa gacgctcgcc accgtcgagg 16740 acctgtgccg caagttcacc accttcggca tcacgcgcca cgacgtcgtc gtctcctgcg 16800 gaggaggctc gacgaccgac accgtcggcc tggcggcggc gctgcaccac cgtggggtgc 16860 cggtggtgca cctgccgacc accctcctgg cccaggtgga cgcgagcgtc ggcggcaaga 16920 cggcggtcaa cctgcccgag ggcaagaacc tcgtcggcgc ctactggcag cccaaggccg 16980 tgctgtgcga caccacgtat ctccagacgc tgcccgccga ggagtgggtc aacggctacg 17040 gcgagatagc gcgctgccac ttcatcggtg ccggcgacct ccgcggcctc gccgtccacg 17100 accaggtcac cgcgagcctg cggctgaagg cgtccgtcgt cgcggccgac gagcgggaca 17160 ccggcctgcg gcacatcctc aactacggcc atacgctggg ccacgcactg gagaccgcca 17220 ccggcttcgg gctgcggcac ggactcggcg tggcgatcgg gacggtcttc gcgggccggc 17280 tcgcggaggc gctgggccgc atcggcgccg accgcgcgcg ggagcacacc gaggtcgtcc 17340 gccactacgg acttcccgac agcctcccgg gaaacaccga catcaccgag ctcgtcgcgc 17400 tgatgaggca cgacaagaag gccacgtcgg gactgacctt cgtgctcgac gggccttccg 17460 gcgtggagct ggtgtccggg atcccggagg acgtcgtcct gcgtacgctc gcggcgatgc 17520 cgcgaggaac ggcctgaccg agtgttccgt cttccgaggg gaagtgaccg tttcgtgtcg 17580 gcagagctgt cagaaccgct gaagaaggcc ctggactccc tggtgttcgg cgtcgtggcg 17640 acgaccgacc ccgacggccg cccgcaccag tcggtggtgt gggtccggcg cgagggctcc 17700 gacgtgctgt tctcgatcac gcgcggcagc cgcaaggaga ggaacatcct gcgcgacccg 17760 cgtgtgagcg tgctgatcag cccggcggac tcgccgtaca cctacgccgc gatccggggc 17820 accgcgcact tcgaggacgt gccggacccg ggcgcgtacc tcgacacgtt ctccataaag 17880 taccacggcg tgccctaccg ggagtcgttc cccgagccgc cggaggtgag caccattctc 17940 gccgtccggc tcgttccgac gtcggtctac gagcagtggt gagggcgtag gcgtcccgaa 18000 gccccggcag cgtcccgaat gccgctgccg gggcttcccg tgggagccct acgccggttt 18060 ccgcgcggtg accaccgagt agccgacctc ctccaccgag cccatgcggt cgatgccgtc 18120 gaccatgcgg tggaacgcct cgtcgtccat gtgggagccg agctcgtccc tggccgcacg 18180 catcttcgcc gccaccgcct cgtaggaggg ccgcacctcg tccccgatgt cgaggaactc 18240 caccacctcc agccccaccg accgcatgca gtcctcgtac gcctcgcggg tgaggacggg 18300 gccctgctgg aagttgtcgt tggcggtgtc g 18331 97 1167 DNA Streptomyces lavendulae 97 atgacaccta cgtccggtga tgacgtcctg tcctttccct catggccgca acacggcgcg 60 gaggagcgcg ccggactcct gcgggccctg gaccagaagg ggtggtggcg cgacgcgggg 120 caggaggtcg atctcttcga gcgggagttc gccgaccacc acggcgcccc gcacgcgatc 180 gccacgacga acggcaccca cgccctggaa ctcgccctgg gggtcatggg gatcggcccc 240 ggtgacgagg tcatcgtccc cgcgttcacc ttcatctcgt cgtcgctggc cgtgcagcgc 300 atgggcgcgg tgccggtgcc ggcggacgta cggcccgaca cctactgcct cgatgccgac 360 gcggcggcgg cgctggtgac gccacgcacc aaagcgatca tgccggtcca catggcgggc 420 cagttcgccg acatggacgc cctggagaag ctctccgtcg cgacgggcgt gccggtcctc 480 caggacgccg cgcacgccca cggcgcgcag tggcagggcc gccgggtcgg ggagctcggc 540 tcgatcgccg ccttcagctt ccagaacggc aagctgatga ccgccggcga gggcggcgcc 600 ctgctcctgc cggacgacga gtccttccac gaggcgttcc tccagcactg ctgcggccgc 660 ccgcccgggg accgcgtcta ccgccatctg acgcagggct ccaactaccg catgaacgag 720 ttctccgcga gcgtcctgcg tgctcaactg aagcgcttga aggatcagtt gcgcatcagg 780 gaggagcgct gggcccagct gcgtacggca ctggccgcca tcgacggcgt ggtgccgcag 840 gggcgcgacg agcgcggcga cctccactcc cactacatgg ccatggtccg gctgcccggc 900 atctcggccc ggcgccgcct cgcgctggtg gacgcgctgg tcgagcgggg agtgcccgcg 960 ttcgtcggct tcccgccggt ctaccgcacc gagggtttcg cgcgcggccc ggcgccggcg 1020 gacgccgagg agctggccaa gagctgtccc gtggcggagg agatcggcag cgactgcctc 1080 tggctgcacc atcgcgtcct gctcgccgac gtgaccacgc tggaccggct ggcggaggtc 1140 ttctccggcc tcgtcggcgc gctctga 1167 98 819 DNA Streptomyces lavendulae 98 gtggtcgtcg tcgacgacaa cgacgggggc gacgccggtg atcaactgat cgccgtgaca 60 ggcgagatga gcggccttct cccgctgcgc gtggtgcggg gaccgctgcg ggggcgggcc 120 gccgcccgga acgccggggc ggccgcggcc ctcgcgcccc ggctggtctt cctcgacgac 180 gacgtcctgg tggggcccgg cttcctcgcc gcacacgccg cggccgcgga accggacgcc 240 ttcacccacg gccggctgcg cgaactcccc accgcggcgc ggttcctcgc cgctgtcgag 300 aaggccgccc cgaccgaggt ccgccgcgcc cgcgccggac tcgaacccgc tgccccggcc 360 gcctccgagc ggcgccaacc gcaccggcgg ctcgtcgcca acgccctgga gcgggccgtg 420 gaggccatgg ccggcggctc cctgccggac gtcgccccct ggctcggctt catcggcgcg 480 aacaccgccc tcgacaaggc cgcatgggag cataccggcg gattcgacga ggagttcggg 540 ctcacctggg ggtgcgagga cctggagttc ggcttccgcc tgcacgccgc cgggctgcgc 600 aggaccctcg cccccgacgc cctcggtgtg cacctcagcc acgcccgccc cggccgctgg 660 gagcagcacc accgcaacct cacgcacttc tccgccggcc acccgcaccc gtcggtacgc 720 gccttggagg ccctgctcgg gcccgacggc acgccggagg cgtatgtgcg cgccgtcctg 780 gccgaagagg ccgcaccggc acgggacgcg gcgcgatga 819 99 783 DNA Streptomyces lavendulae 99 atgagcggca caccggccac cgcgccgtac ggtcccgtgg tgctctcccc gcacgcggac 60 gacgccgtgt ggtccctggg cgggcggctg gcgcgctggg ccgccgaggg cccgcggccg 120 accgtcgtca cggtcttcgc cgggcccgcg gccgggaagc ccgagtcgtg gcggagcgcc 180 gccgatcccg cggtgcgccg ggccgaggac cgggcggcat gtgccgaact gggcgtgcgc 240 cacgtgccgc tgggcttcac cgacgcggca ctgcgtacgg cctcgggcgc ctatctctac 300 gcttccccgc gccggctctt cggcccctgg cacccggccg acctcccgct gctggaggag 360 gtgcgggcgg ctctgctgcc gctgtgcgcg ggggcgtcga gcgtccacgt tcccctggcg 420 gcgggccggc acgtcgacca ccgcctggtc cgcggcgcgg tggagcccct gtcccccgcc 480 cgtaccgtct tctacgagga cttcccctac cggctgcgcg aacgtgacca cacgaacctg 540 cggccgcgca cggaacggct gccgtccgag gcggtggacc gctggctgac cgccgccggt 600 cactactcca gccaggcgag cgcccacttc ggcggtgcgg ccgccctgcg cgaggccctg 660 ttcgcccgcg cccgcgcaca cggcgggccc ggccggcccg gccacgccga ccgccactgg 720 gtgcccgtcg gccaggacga ccggggcgag gcccggccgg cacccgtgga aagggggccg 780 tga 783 100 383 PRT Streptomyces lavendulae 100 Met Ser Arg Ser Thr His Pro Pro Thr Ala Thr Pro Asp Ala Gly Thr 1 5 10 15 Arg Arg Arg Leu Pro Leu Ile Gly Asn Asp Leu Val Ile Asn Glu Asp 20 25 30 Ser Cys Asn Leu Ser Cys Thr Tyr Cys Leu Thr Gly Gln Ser Asn Leu 35 40 45 Lys Glu Gly His Ser Leu Gln Leu Ile Phe Glu Pro Pro Arg Arg Asp 50 55 60 Ser Tyr Ala Lys Asp Ser Gly Leu Gly Gln Arg Met Asp Lys Val Ala 65 70 75 80 Asp Arg Ile Arg Asp Arg Phe Gly Leu Pro Leu Leu Lys Val Thr Gly 85 90 95 Gly Glu Ile Phe Leu Val Arg Gly Ile Met Asp Phe Leu Glu Gln Glu 100 105 110 Ala Arg Lys Tyr Asp Val Leu Val Ile Gln Thr Asn Gly Val Leu Val 115 120 125 Arg Glu Glu His Leu Glu Arg Phe Arg Ser Trp Gly Asn Val Val Leu 130 135 140 Gln Val Ser Leu Asp Ser His Leu His His Gly Asn Ser His Arg Val 145 150 155 160 Pro Ser Gly Ser Leu His Glu Lys Val Val Ala Ala Ile Ala Arg Ile 165 170 175 Leu Asp Ser Gly Leu Pro Val Glu Ile Tyr Ser Val Leu Asn Asp Arg 180 185 190 Ser Val Thr Glu Val Cys Ala Phe Ala Glu Trp Leu Ser Gly Phe Ser 195 200 205 Arg Pro Pro Val Tyr Phe Pro Phe Pro Val Arg Gly Pro Asp Ser Glu 210 215 220 Asp Phe Lys Val Arg Pro Gly Gln Phe Gly His Ile Gln Glu Leu Val 225 230 235 240 Asp Arg Tyr Asp Glu Phe Ala Arg Val Leu Pro Pro Arg Pro Tyr Phe 245 250 255 Asp Arg Leu Thr Ser Phe Tyr Arg Glu Gly Arg Arg Thr Phe Arg Cys 260 265 270 His Leu Pro Arg Leu Val Val Ser Ser Phe Ser Asp Gly Val Val Thr 275 280 285 Pro Cys Pro Asn Ile Trp Phe Ser Asp Met Gly Asn Ala Leu Glu Asp 290 295 300 Asp Trp Ser Glu Met Leu Asp Thr Val Gly Thr Ser Gly Leu Tyr Arg 305 310 315 320 Ala Leu Leu Ala Pro Lys Pro Arg Leu Lys Ala Cys His Gly Cys Phe 325 330 335 Thr Pro Trp Asp Thr Leu Ser Met Tyr Phe Glu Asp Glu Ile Thr Leu 340 345 350 Asp Glu Leu Cys Ala Ala Pro Thr Tyr Ser Pro Pro Arg Ile Arg Gln 355 360 365 Met Leu Ser Asp Ala Lys Ala Asp Tyr Leu Gln Gly Gly His Asp 370 375 380 101 707 PRT Streptomyces lavendulae 101 Met Ala Leu Arg Ala Pro Asn Ser Pro Arg Trp Val Val Ala Phe Leu 1 5 10 15 Ser Leu Leu Ala Ser Gly Ala Arg Pro Leu Leu Leu Glu Pro Asp Thr 20 25 30 Pro Gly Pro Glu Thr Ala Arg Leu Leu Arg Ala Ala Gly Gly Gly Arg 35 40 45 Ser Leu Val Val Pro Gly Thr Gly Asp Gly Leu Arg Leu Thr Leu Thr 50 55 60 Gly Ser Pro Gly Glu Pro Ser Gly Ala Pro Pro Ala Val Leu Leu Pro 65 70 75 80 Thr Ser Gly Ser Thr Gly Ala Ser Lys Leu Val Ala Arg Ser Glu Glu 85 90 95 Ser Leu Leu Ala Glu Gly Arg Arg Tyr Arg Asp Gly Val Gly Leu Thr 100 105 110 Gly Glu Asp Thr Leu Leu Leu Pro Val Pro Leu Ser His Ala Tyr Ala 115 120 125 Leu Gly Trp Leu Phe Gly Gly Leu Leu Thr Gly Ala Ala Leu Arg Pro 130 135 140 Val Pro Pro Thr Ala Leu Gly Arg Ile Ala Ala Glu Leu Ser Gly Gly 145 150 155 160 Ala Thr Val Val Ala Leu Val Pro Ser Val Ala Arg Leu Leu Ala Thr 165 170 175 Arg Arg Leu Arg Gly Ala Ala Ala Gly Arg Ala Pro Ala Ala Pro Gly 180 185 190 Leu Arg Leu Ala Met Val Gly Ala Gly Pro Val Asp Glu Gln Leu Asp 195 200 205 Arg Ala Phe Thr Glu Ala Phe Gly Thr Gly Leu Ala Arg Asn Tyr Gly 210 215 220 Ser Thr Glu Thr Gly Ala Val Leu Ala Gly Pro Ala Gly Leu Glu Pro 225 230 235 240 Leu Cys Ala Gly Ala Pro Leu Pro Gly Val Glu Cys Glu Leu Thr Gly 245 250 255 Pro Glu Gly Val Val Pro Pro Ala Gly Thr Pro Gly Leu Leu Ser Val 260 265 270 Arg Val Asp Gly Arg Pro Tyr Ala Met Gly Asp Leu Ala Val Ala Val 275 280 285 Pro Gly Gly Leu Arg Ile Leu Gly Arg Glu Asp Arg Ala Ile Arg Arg 290 295 300 Gly Gly Arg Trp Val Ser Pro Leu Glu Ile Glu Glu Val Leu Arg Gly 305 310 315 320 His Pro Asp Val Val Asn Val Arg Val Gly Ala Arg Arg Gly Arg His 325 330 335 Arg Gly Glu Asp Gly Ile Val Ala Glu Val Ser Ala Ala Gly Pro Gly 340 345 350 Leu Thr Pro Glu Ala Leu Arg Glu His Ala Arg Arg Glu Leu Ala Pro 355 360 365 His Lys Val Pro Asp Glu Phe Val Leu Arg Glu Ser Leu Pro Val Asn 370 375 380 Ala Ala Gly Lys Val Arg Ala Ala Ser Val Tyr Arg Leu Thr Arg Ser 385 390 395 400 Ala Ala Glu Ala Ala Arg Ala Tyr Lys Ala Ser Glu Val Leu Phe Ala 405 410 415 Leu His Asp Leu Gly Ala Leu Glu Ala Leu Ala Gln Gly Ala Gly Thr 420 425 430 Ala Leu Leu Ala Gly Glu Leu Gly Cys Asp Ala Asp Ala Leu Glu Trp 435 440 445 Leu Leu Arg Thr Ala Thr Ala Leu Gly Val Leu Thr Thr Gly Ala Gln 450 455 460 Glu Pro Gly Asp Arg Val Arg Ala Gly Glu Leu Ala Ala Phe Val Ala 465 470 475 480 Leu Glu Glu His Leu Ser Arg Gly Leu Val Thr Arg Glu Glu Leu Val 485 490 495 Ala Val Ala Arg Ser Gly Thr Ala Arg Arg Pro Phe Glu Glu Arg Pro 500 505 510 Pro Glu Ser Leu Gly Pro Leu Val Ala Leu Tyr Gln Gly Ala Met Asp 515 520 525 Gly Pro Gly Ala Arg Ala Arg Ala Ala Leu Gly Leu Arg Leu Leu Arg 530 535 540 Pro Gly Pro Gly Ala Arg Val Val Glu Val Thr Ala Gly Pro Gly Arg 545 550 555 560 Tyr Leu Glu Arg Leu Leu Ala Ser Asp Pro Gly Ala Ser Gly His Leu 565 570 575 Val Thr Val Gly Arg Leu Ser Gly Pro Leu Ser Ser Ala Val Ala Ala 580 585 590 Ala Val Glu Glu Gly Arg Val Thr Val Gly Thr Glu Leu Pro Val Gly 595 600 605 Tyr Ala Asp Phe Cys Val Val Ala Asn Ala Val His Gly Pro Gly Pro 610 615 620 Gly Ser Ala Leu Gly Ala Leu Leu Gly Ser Leu Arg Pro Gly Gly Arg 625 630 635 640 Leu Leu Val Asp Asp Val Phe Leu Pro Ala Ser Gly Pro Gly Ser Glu 645 650 655 Leu Ala Leu Asp Trp Leu Thr His Gly Gly Thr Ala Trp Pro Ala Thr 660 665 670 Gly Glu Leu Ile Ala Gly Leu Leu Gln Glu Gly Ala Glu Val Ala Arg 675 680 685 His Val Pro Leu Asp Ala Ser Pro Cys His Leu Ile Ile Ala Lys Glu 690 695 700 Ala Gly Ser 705 102 257 PRT Streptomyces lavendulae 102 Met Ser Thr Val Thr Asp Arg Ala Thr Glu Arg Leu Gly Gln Ser Gly 1 5 10 15 Arg Val Val Val Val Ser Gly Ala Ser Gly Gln Ile Gly Gly Ala Cys 20 25 30 Ala Leu Glu Leu Ala Ala Leu Gly Ala Thr Val Val Ala Gly Tyr His 35 40 45 Ser Gly Glu Gln Ala Ile Arg Lys Leu Arg Glu Gln Val Glu Gly Gln 50 55 60 Gly Gly Thr Leu Val Pro Val Ala Ala Asp Leu Ser Glu Pro Glu Gly 65 70 75 80 Ala Asp Ala Leu Val Ala Ala Ala Val Glu Arg Phe Gly Arg Val Asp 85 90 95 Gly Cys Val Ala Ala Ala Gly Leu Arg Thr Arg Arg Leu Ala Met Ala 100 105 110 Thr Asp Ala Arg Ser Leu Glu Lys Leu Leu Arg Val Asn Leu Ala Gly 115 120 125 Ser Val Gly Leu Ala Lys Ala Cys Leu Lys Pro Met Met Arg Ala Arg 130 135 140 Tyr Gly Arg Ile Val Leu Phe Gly Ser Arg Ala Gly Thr Ser Gly Leu 145 150 155 160 Pro Gly His Ser Ala Tyr Ala Ala Thr Lys Gly Ala Leu Gln Pro Trp 165 170 175 Ala Ala Ser Val Ala Gly Glu Val Gly Lys His Gly Ile Thr Val Asn 180 185 190 Val Val Ala Pro Gly Ala Ile Arg Ala Glu Val Met Asp Phe Ser Glu 195 200 205 Ala Glu Arg Asp Leu Val Leu Gln Phe Ile Gly Ala Gly Arg Leu Gly 210 215 220 Glu Pro Glu Glu Val Ala Ala Ala Val Ser Phe Leu Leu Ser Pro Ser 225 230 235 240 Ala Ser Tyr Val Asn Gly Asn Thr Leu Val Val Asp Gly Gly Ala Arg 245 250 255 Phe 103 404 PRT Streptomyces lavendulae 103 Met Thr Pro Pro Thr Thr Ala Arg Glu Pro Leu Arg Met Ala Val Leu 1 5 10 15 Gly Ala Gly Trp Val Ser Arg Lys Val Trp Leu Pro Leu Leu Ala Glu 20 25 30 His Pro Ala Phe Arg Val Asp Phe Leu Val Asp Asp Asp Pro Val Ala 35 40 45 Ala Arg Ser Ala Leu Pro Glu Gly Ala Arg Thr Arg Val Leu Ser Arg 50 55 60 Pro Glu Glu Leu Ala Pro Arg Ser Val Asp Ala Ala Ile Ile Ala Leu 65 70 75 80 Pro Asn His Leu His Leu Pro Val Ala Lys Ala Leu Leu Glu Arg Asp 85 90 95 Val Pro Val Phe Val Glu Lys Pro Val Cys Arg Thr Leu Phe Glu Ala 100 105 110 Gln Ala Leu Ala Leu Asp His Gln Ala Arg Gly Asp Ser Ile Gly Asp 115 120 125 Ile Thr Leu Tyr Ala Trp Ser Ala Ala Arg His Arg Thr Asp Val Cys 130 135 140 Arg Leu Ala Glu Leu Leu Pro Ser Leu Gly Thr Val Arg Ser Val Gly 145 150 155 160 Leu Ser Trp Ile Arg Ala Thr Gly Ile Pro Gln Arg Thr Gly Trp Phe 165 170 175 Val Asp Arg Arg Leu Ala Gly Gly Gly Ala Leu Leu Asp Leu Gly Trp 180 185 190 His Leu Leu Asp Val Gly Leu His Leu Leu Gly Trp Pro Arg Val Val 195 200 205 Arg Ala Ala Ser Thr Met Ser Ala Asp Trp Met Ser Arg Gly Glu Ala 210 215 220 Thr Ala Asp Trp Ser Arg Arg Ser Ser Gly Thr Ala Arg Pro Gly Pro 225 230 235 240 Gly Glu Thr Val Glu Asp Thr Ala Arg Gly Phe Leu Val Thr Asp Thr 245 250 255 Asp Val Gly Ile Ser Leu Glu Thr Arg Trp Ala Ser His Gln Ala Leu 260 265 270 Asp Val Thr Thr Ile Thr Val Glu Gly Thr Glu Gly Val Ala Thr Leu 275 280 285 Arg Gly Thr Phe Gly Phe Ser Pro His Arg Leu Gln Lys Ser Ser Leu 290 295 300 Val Val Leu Arg Gln Gly Val Glu Glu Thr Val Ala Leu Pro Asp Glu 305 310 315 320 Pro Val Gly Val Glu Tyr Arg Arg Gln Val Asp Glu Leu Ala Arg Arg 325 330 335 Leu Gly Gly Ser Ala Asp Gly Gln Gly Pro Val Ser Gly Leu Gly Glu 340 345 350 Gly Ser Met Ala Glu Val Thr Ile Leu Ala Ser Cys Ile Asp His Ile 355 360 365 Tyr Ser Ala Ala Gly Val Asp Pro Pro Ser Pro Leu His Arg Pro Gln 370 375 380 Ser Asp Ala Ala Pro Ser Thr Ser Ser Cys Pro Arg Val Leu Pro Thr 385 390 395 400 Arg Gly Ser Gln 104 382 PRT Streptomyces lavendulae 104 Met Lys Phe Ala Tyr Phe Ser His Val Trp Gly Arg Pro Gly Ile Thr 1 5 10 15 Pro Gly Glu Arg Tyr Glu Glu Leu Trp Arg Glu Val Glu Asp Ala Asp 20 25 30 Arg Leu Gly Phe Asp Tyr Ala Phe Ser Val Glu His His Cys Thr Pro 35 40 45 His Glu Ser Trp Met Pro Ser Pro Ala Val Phe Cys Thr Gly Ala Ala 50 55 60 Leu Arg Thr Glu Arg Ile Arg Val Gly Pro Met Gly Trp Val Pro Pro 65 70 75 80 Leu Arg His Pro Leu His Leu Val Glu Glu Val Ala Thr Leu Asp Gln 85 90 95 Leu Leu Gly Gly Arg Leu Glu Val Gly Leu Ala Ser Gly Val Ser Arg 100 105 110 Asp Pro Phe Leu Pro Phe Asp Ala Asp Phe Asp Asn Arg His Leu Leu 115 120 125 Thr Arg Glu Ala Leu Glu Leu Leu Arg Ala Ala Phe Ala Ala Arg Gly 130 135 140 Ala Phe Asp Phe Asp Gly Pro Ala His Arg Leu Arg Asp Ile Ala Leu 145 150 155 160 Ser Phe Pro Pro Val Gln Arg Pro His Pro Pro Met Trp Val Pro Thr 165 170 175 Thr Asn Arg Asn Thr Leu Arg Tyr Leu Ser Glu Ala Gly Ala His Thr 180 185 190 Ser Ser Thr Met Ile Val Pro Arg Ala Ser Met Ala Leu Val Tyr Arg 195 200 205 His Tyr Leu Asp Trp Trp Arg Gly His Gly His Ala Ser Asp Pro Arg 210 215 220 Ile Gly Tyr Trp Thr Leu Val His Val Ala Arg Thr Asp Ala Glu Ala 225 230 235 240 Glu Glu Arg Ala Ala Ala His Ile Thr Glu Thr Phe Thr Lys Thr Leu 245 250 255 Arg Tyr Gly Ser Val Ser Arg Ser Arg Asp Gln His Ala Pro Pro Ser 260 265 270 Arg Leu Ser Thr Thr Asp Ile Leu Ala Gly Ser Gly Asp Leu Arg Phe 275 280 285 Leu Leu Glu Asn Asn Leu Val Phe Val Gly Ser Pro Ala Thr Val Ala 290 295 300 Asp Arg Ile Arg Ala Ala Ser Leu Glu Gly His Phe Asp Thr Leu Leu 305 310 315 320 Gly Glu Phe Thr Phe Gly Glu Leu Ala Asp Arg His Arg Ile Glu Ser 325 330 335 Met Glu Leu Phe Ala His Glu Val Ala Pro Ala Leu Arg Ala Phe Ser 340 345 350 Pro Tyr Ala Pro Arg Pro Gln Glu Pro Ala Tyr Thr Ala Ser Asp Glu 355 360 365 Gln Gln Val Ala Ala Arg Leu Gln Ala Leu Gly Tyr Ile Asp 370 375 380 105 290 PRT Streptomyces lavendulae 105 Met Glu Arg Leu Lys Leu Val Pro Asp Glu His Arg Arg Phe Thr Val 1 5 10 15 Asp Glu Gln Ser Ala Arg Arg Leu His Arg Ile Gly Pro Glu Leu Leu 20 25 30 Ser Ala Leu Cys Glu Ala Gly Val Pro Phe Val Gly Ser Gly Ala Gly 35 40 45 Arg Leu Phe Asp Gly Tyr Asp Leu Gly Asn Ala Ala Leu His Leu Gly 50 55 60 Leu Ser Ser Val Gln Arg Arg Ala Ile Arg Ser Trp Ala Gly Ser Leu 65 70 75 80 Arg Thr Ala Ser Ala Ala Glu Ser Pro Arg Trp Arg Val Asp Val Thr 85 90 95 Ala Ser Cys Pro Val Pro Gly His Ala Gly Pro Cys Arg Tyr Gly Val 100 105 110 Leu Leu Pro Gly Ala Arg Arg Pro Val Glu Ala Ala Ser Pro Arg Glu 115 120 125 Thr Thr Leu Ala Arg Leu Tyr Thr Arg Ser Arg Gly His Trp Pro Asp 130 135 140 Phe Pro Pro Ala Val Leu Asp Leu Leu Arg Thr Leu Glu Pro Val Gly 145 150 155 160 Phe Phe Leu Leu Pro Glu Ala Ile Arg Trp Asp Pro Gly Phe Leu Trp 165 170 175 Ser Thr His Met Ala Asp Cys Gly Gly Ala Ala Ala Trp Leu Val Ala 180 185 190 Glu Gly Arg Arg Arg Gly Leu Asp Val Arg Phe Ser Phe Gly Leu Leu 195 200 205 Val Ala Lys Pro Tyr Ser Thr Pro His Cys Trp Ala Glu Phe Leu Val 210 215 220 Gly Gly Arg Trp Val Pro Ala Asp Pro Leu Leu Leu Arg Ala Met Ala 225 230 235 240 Ala Trp Gly Gly Leu Asp Ala Ala Ala His Pro Pro His Ser Ser Pro 245 250 255 Gly Ala Val Tyr His Arg Leu Ala Gly Arg Phe Thr Lys Val Val Ser 260 265 270 His Ala Gly Val Trp Ala Pro Thr Ser Leu Pro Thr Glu Leu Leu Pro 275 280 285 Cys Pro 290 106 235 PRT Streptomyces lavendulae 106 Met Pro Leu Asn Pro Pro Pro Ala Ser Arg Ala Ala Ala Asp Ala Pro 1 5 10 15 Ala Thr Ala Leu Pro Cys Arg Phe Thr Thr Val Val Phe Asp Leu Asp 20 25 30 Gly Val Leu Ile Asp Ser Phe Ala Val Met Arg Glu Ala Phe Ala Val 35 40 45 Ala Tyr Arg Glu Val Val Gly Pro Gly Glu Pro Pro Phe Glu Glu Tyr 50 55 60 Arg Thr His Gln Gly Arg Tyr Phe Pro Asp Ile Met Arg Leu Met Gly 65 70 75 80 Leu Pro Gly Glu Met Glu Glu Pro Phe Val Arg Glu Ser His Arg Leu 85 90 95 Met Asp Arg Val Glu Val Tyr Pro Asp Val Pro Gln Leu Leu Ala Glu 100 105 110 Leu Arg Ala Asp Gly Val Gly Thr Ala Ile Ala Thr Gly Lys Ser Gly 115 120 125 Ser Arg Ala Arg Ala Val Leu Glu Ala Val Gly Leu Leu Pro Leu Leu 130 135 140 Asp Glu Val Val Gly Ser Asp Glu Val Pro Arg Pro Lys Pro His Pro 145 150 155 160 Asp Ile Val Arg Glu Ala Leu Arg Arg Leu Asp Ala Ala Pro Glu Asp 165 170 175 Ala Val Met Val Gly Asp Ala Val Ile Asp Ile Arg Ser Gly Arg Ala 180 185 190 Ala Gly Thr Ala Thr Val Gly Ala Thr Trp Gly Glu Gly Ala Ala Gly 195 200 205 Gln Leu Arg Ala Glu Arg Pro Asp Phe Leu Leu Asp Lys Pro Gln Ser 210 215 220 Leu Leu Ala Leu Val Arg Ser Gly Gly His Ala 225 230 235 107 346 PRT Streptomyces lavendulae 107 Met Thr Pro Ala Thr Pro Arg Trp Ser Val Val Ala Pro Gln Gly Thr 1 5 10 15 Asn Leu Glu Leu Ala Gly Thr Gly Gly Arg Glu Gly Trp Arg Leu Leu 20 25 30 Leu Glu Thr Ala Arg Thr Val His Arg His Gly Arg Gly Ala Leu Trp 35 40 45 Leu Leu Asp Arg Thr Asp Thr Leu Pro Arg Arg Glu Pro Glu Pro Val 50 55 60 Trp Glu Gly Trp Thr Ala Leu Ala Ala Leu Ala Gly Ala Val Pro Gly 65 70 75 80 Leu Asp Leu Gly Leu Leu Ser Ser Ala Pro Pro Phe Arg Asn Ala Ala 85 90 95 Leu Ile Ala Lys Arg Ala Ala Thr Leu Asp Val Val Cys Asp Gly Arg 100 105 110 Leu Thr Leu Gly Phe Pro Ala Arg Glu Tyr Leu Pro Glu His His Ser 115 120 125 Thr Gly Arg Glu Val Pro Thr Gly Leu Glu Ala Asp Glu Glu Glu Ala 130 135 140 Ala Gly His Arg Ala Leu Gly Glu Thr Val Glu Ala Leu Arg Ala Leu 145 150 155 160 Trp Gly Gly Gln Pro Val Thr Phe Thr Gly Glu His Ile Arg Leu Thr 165 170 175 Ser Ala His Cys Val Pro Ala Pro Arg Gln Gln Pro Leu Pro Leu Ala 180 185 190 Leu Arg Thr Pro Ala Gly Asp Ala Gly Ser Gly Ala Leu Arg Pro Ala 195 200 205 Asp Ala Thr Val Arg Glu Cys Ala His Val Gln Trp Thr Gly Glu Pro 210 215 220 Ala Gln Val Ala Ala Ala Val Thr Ala Phe Arg Arg Arg Arg Thr Glu 225 230 235 240 Leu Gly Leu Asp Pro Asp Gly Val Arg His Ala Trp Ala Ala Glu Cys 245 250 255 Arg Ile Phe Asp Ser Val Leu Glu Arg Asp Arg Trp Leu Ser Thr Pro 260 265 270 His Glu Val Leu Phe Trp Ser His His Pro Asp Leu Leu Ala Arg Arg 275 280 285 Ser Leu Tyr Gly Thr Pro Glu Gln Leu Thr Glu Arg Ala Arg Arg Leu 290 295 300 Val Ala Ala Gly Val Ala Glu Phe Val Leu Trp Phe Arg Asp Tyr Pro 305 310 315 320 Ala Thr Thr Ser Leu Glu Arg Leu Phe Gln Glu Val Val Pro Gln Val 325 330 335 Ala Pro Gly Ala Ala Lys Glu Ala Glu Glu 340 345 108 520 PRT Streptomyces lavendulae 108 Met Leu Asn Thr Leu Ser Thr Ala Pro Phe Leu Ser Thr Ala Trp Leu 1 5 10 15 Ala Gly Ala Ala Arg Leu Glu Arg Pro Pro Val Gly Glu Arg Gly Thr 20 25 30 Val Ala Leu Arg Leu Glu Leu Thr Asp Pro Pro Pro Gly Glu Pro Pro 35 40 45 Ala Val Asp Val Gln Val Asp Leu Val Ala Gly Arg Leu Gly Leu Ala 50 55 60 Ala Ala Ala Gly Glu Ser Pro Gly Leu Arg Ile Arg Leu Pro Leu Glu 65 70 75 80 Ala Ala Arg Ala Leu Leu Leu Gly Pro Ala Arg Asp Arg Thr Gly Val 85 90 95 Phe Glu Arg Gly Asp Val Arg Ala Glu Gly Asn Phe Ser Leu Leu Phe 100 105 110 Phe Ile Asp Ala Ala Leu Glu Arg Asp Ala Ser Gly His Val Ala Ala 115 120 125 Leu Arg Gly Thr Pro Gly Thr Thr Ala Arg Glu Ala Ala Pro Pro Pro 130 135 140 Gly Thr Glu Asp Ala Ala Glu Ala Val Arg Arg Ala Arg Ala Ala Leu 145 150 155 160 Pro Gly Thr Met Arg Glu Leu Glu Arg Glu Val Gly Thr Ser Thr Pro 165 170 175 Gly Ala Gln Ile Tyr Val Ser Arg Asp Gly Val Pro Leu Ala Asp Ala 180 185 190 Gly Leu Gly Leu Ala Arg Pro Gly Val Ala Met Thr His Arg Ser Leu 195 200 205 Pro Leu Trp Tyr Cys Cys Ala Lys Pro Leu Leu Ser Val Ala Leu Gly 210 215 220 Arg Leu Trp Glu Ala Gly Ala Tyr Asp Pro Tyr Leu Pro Val Ala His 225 230 235 240 Tyr Leu Pro Glu Phe Gly Asn Arg Gly Lys Glu Ser Ile Thr Ser Met 245 250 255 Glu Leu Leu Thr His Thr Gly Pro Leu Pro Thr Gly Asp Asp Pro Leu 260 265 270 His Gly Ile Val Ala Gly Pro Asp Glu Glu Arg Val Arg Arg Ala Phe 275 280 285 Glu Val Pro Val Ala Pro Arg Pro Gly Gly Thr Pro Gly Ile Asn Tyr 290 295 300 Ser Gln Trp Trp Ala Trp Phe Val Leu Ala Arg Ile Leu Pro Val Val 305 310 315 320 Asp Gly Arg Glu Tyr Arg Ala Tyr Val Gln Glu Glu Ile Leu Gly Pro 325 330 335 Cys Gly Met Ser Gly Thr Arg Val His Leu Asp Arg Glu Glu Phe Ala 340 345 350 Ala Leu Gly Gly Glu Leu Pro Leu Ile His Val Ser Asn Pro Glu Gly 355 360 365 Gly Pro Leu Pro Thr His Trp Trp Ser Thr Glu Ala Ala Thr Thr Arg 370 375 380 Cys Ile Pro Gly Val Asn Thr Arg Gly Pro Leu Arg Asp Met Gly Arg 385 390 395 400 Leu Phe Glu Met Leu Leu Arg Gly Gly Asp Ala Pro Gly Gly Arg Val 405 410 415 Leu Ala Pro Pro Thr Val Ala Ala Leu Thr Ala Arg His Arg Thr Gly 420 425 430 Leu Gln Asp Arg Tyr Gly Asn Ala Asp Trp Gly Met Gly Phe Arg Leu 435 440 445 Glu Cys Arg Gln Leu Asp Pro Arg Phe Thr Ser Phe Gly Ser Tyr Ala 450 455 460 Ser Pro Arg Ser Phe Gly His Asp Gly Leu Trp Thr Ala Val Val Phe 465 470 475 480 Ala Asp Pro Asp Ala Ala Leu Val Val Ala Leu His Leu Asn Gly Lys 485 490 495 Val Glu His Glu Arg His Arg Glu Arg Ile Val Arg Leu Ala Asp Ala 500 505 510 Val Tyr Gln Asp Leu Arg Leu Ser 515 520 109 283 PRT Streptomyces lavendulae 109 Met Pro His Ser Glu Leu Ser Glu Leu Pro Met Pro Ser Pro Ala Ser 1 5 10 15 Glu Glu Val Gly Ala Leu Tyr Asp Arg Phe Thr Ala Leu Gly Ala Ala 20 25 30 Ser Leu Gly Glu Asn Leu His Phe Gly Tyr Trp Asp Ser Pro Asp Ser 35 40 45 Gln Val Pro Leu Ala Glu Ala Thr Asp Arg Leu Thr Asp Met Met Ala 50 55 60 Glu Arg Leu Arg Ile Gly Ala Gly Ser Arg Val Leu Asp Leu Gly Cys 65 70 75 80 Gly Val Gly Thr Pro Gly Val Arg Ile Ala Arg Leu Ser Gly Ala His 85 90 95 Val Thr Gly Ile Ser Val Ser His Glu Gln Val Val Arg Ala Asn Ala 100 105 110 Leu Ala Glu Glu Ala Gly Leu Ala Asp Arg Ala Arg Phe Gln Arg Ala 115 120 125 Asp Ala Met Asp Leu Pro Phe Glu Asp Glu Ser Phe Asp Ala Val Ile 130 135 140 Ala Leu Glu Ser Ile Ile His Met Pro Asp Arg Ala Gln Val Leu Ala 145 150 155 160 Gln Val Gly Arg Val Leu Arg Pro Gly Gly Arg Leu Val Leu Thr Asp 165 170 175 Phe Phe Glu Arg Ala Pro Leu Ala Pro Glu Gly Arg Ala Ala Val Gln 180 185 190 Arg Tyr Leu His Asp Phe Met Met Thr Met Val Ser Ala Glu Ala Tyr 195 200 205 Pro Pro Leu Leu Arg Gly Ala Gly Leu Trp Leu Glu Glu Phe Leu Asp 210 215 220 Ile Ser Asp Gln Thr Leu Glu Lys Thr Phe Arg Leu Leu Ser Glu Arg 225 230 235 240 Ile Asn Ser Ser Lys Gln Arg Leu Glu Thr Gln Phe Gly Glu Glu Met 245 250 255 Val Asn Gln Phe Asp Pro Gly Asp Leu Val Gly Val Lys Glu Phe Gly 260 265 270 Tyr Leu Leu Leu Val Ala Gln Arg Pro Gly Lys 275 280 110 275 PRT Streptomyces lavendulae 110 Met Thr Glu Thr Ala Ser Ala Ser Asp Arg Met Val Glu Leu Tyr Asn 1 5 10 15 Arg Val Thr Asp Leu Met Val His Ala Glu Gly Gly Tyr Met His Gly 20 25 30 Gly Tyr Trp Ala Gly Pro Asp Val Pro Thr Thr Val Glu Glu Ala Gly 35 40 45 Asp Arg Leu Thr Asp Tyr Val Ser Glu Arg Leu Arg Leu Ala Pro Gly 50 55 60 Glu Arg Val Leu Asp Val Gly Ser Gly Asn Gly Lys Ala Thr Leu Arg 65 70 75 80 Ile Ala Ala Arg His Gly Val Arg Ala Thr Gly Val Ser Ile Asn Pro 85 90 95 Tyr Gln Val Gly Leu Ser Arg Gln Leu Ala Glu Lys Glu Gly Asp Glu 100 105 110 Ala Thr Glu Phe Arg Ile Gly Asp Met Leu Ala Leu Pro Phe Pro Asp 115 120 125 Gly Ser Phe Asp Ala Cys Tyr Ala Ile Glu Ser Ile Cys His Ala Leu 130 135 140 Glu Arg Ala Asp Val Phe Thr Glu Ile Ala Arg Val Leu Arg Pro Gly 145 150 155 160 Gly Arg Val Thr Val Thr Asp Phe Val Leu Arg Arg Pro Leu Ser Asp 165 170 175 Ala Ser Arg Thr Ile Val Asp Thr Ala Asn Asp Asn Phe Gln Gln Gly 180 185 190 Pro Val Leu Thr Arg Glu Ala Tyr Glu Asp Cys Met Arg Ser Val Gly 195 200 205 Leu Glu Val Val Glu Phe Leu Asp Ile Gly Asp Glu Val Arg Pro Ser 210 215 220 Tyr Glu Ala Val Ala Ala Lys Met Arg Ala Ala Arg Asp Glu Leu Gly 225 230 235 240 Ser His Met Asp Asp Glu Ala Phe His Arg Met Val Asp Gly Ile Asp 245 250 255 Arg Met Gly Ser Val Glu Glu Val Gly Tyr Ser Val Val Thr Ala Arg 260 265 270 Lys Pro Ala 275 111 146 PRT Streptomyces lavendulae 111 Met Phe Arg Leu Pro Arg Gly Ser Asp Arg Phe Val Ser Ala Glu Leu 1 5 10 15 Ser Glu Pro Leu Lys Lys Ala Leu Asp Ser Leu Val Phe Gly Val Val 20 25 30 Ala Thr Thr Asp Pro Asp Gly Arg Pro His Gln Ser Val Val Trp Val 35 40 45 Arg Arg Glu Gly Ser Asp Val Leu Phe Ser Ile Thr Arg Gly Ser Arg 50 55 60 Lys Glu Arg Asn Ile Leu Arg Asp Pro Arg Val Ser Val Leu Ile Ser 65 70 75 80 Pro Ala Asp Ser Pro Tyr Thr Tyr Ala Ala Ile Arg Gly Thr Ala His 85 90 95 Phe Glu Asp Val Pro Asp Pro Gly Ala Tyr Leu Asp Thr Phe Ser Ile 100 105 110 Lys Tyr His Gly Val Pro Tyr Arg Glu Ser Phe Pro Glu Pro Pro Glu 115 120 125 Val Ser Thr Ile Leu Ala Val Arg Leu Val Pro Thr Ser Val Tyr Glu 130 135 140 Gln Trp 145 112 343 PRT Streptomyces lavendulae 112 Met Gln Gln Pro His His Ser Arg Val Asp Val Glu Leu Gly Glu Arg 1 5 10 15 Ser Tyr Pro Val His Val Gly Pro Gly Val Arg His Leu Leu Pro Gly 20 25 30 Ile Val Ala Ser Leu Gly Ala His Arg Ala Ala Val Val Thr Ala Arg 35 40 45 Pro Pro Asp Leu Val Pro Asp Pro Gly Val Pro Ala Leu Ile Val Arg 50 55 60 Ala Arg Asp Gly Glu Arg His Lys Thr Leu Ala Thr Val Glu Asp Leu 65 70 75 80 Cys Arg Lys Phe Thr Thr Phe Gly Ile Thr Arg His Asp Val Val Val 85 90 95 Ser Cys Gly Gly Gly Ser Thr Thr Asp Thr Val Gly Leu Ala Ala Ala 100 105 110 Leu His His Arg Gly Val Pro Val Val His Leu Pro Thr Thr Leu Leu 115 120 125 Ala Gln Val Asp Ala Ser Val Gly Gly Lys Thr Ala Val Asn Leu Pro 130 135 140 Glu Gly Lys Asn Leu Val Gly Ala Tyr Trp Gln Pro Lys Ala Val Leu 145 150 155 160 Cys Asp Thr Thr Tyr Leu Gln Thr Leu Pro Ala Glu Glu Trp Val Asn 165 170 175 Gly Tyr Gly Glu Ile Ala Arg Cys His Phe Ile Gly Ala Gly Asp Leu 180 185 190 Arg Gly Leu Ala Val His Asp Gln Val Thr Ala Ser Leu Arg Leu Lys 195 200 205 Ala Ser Val Val Ala Ala Asp Glu Arg Asp Thr Gly Leu Arg His Ile 210 215 220 Leu Asn Tyr Gly His Thr Leu Gly His Ala Leu Glu Thr Ala Thr Gly 225 230 235 240 Phe Gly Leu Arg His Gly Leu Gly Val Ala Ile Gly Thr Val Phe Ala 245 250 255 Gly Arg Leu Ala Glu Ala Leu Gly Arg Ile Gly Ala Asp Arg Ala Arg 260 265 270 Glu His Thr Glu Val Val Arg His Tyr Gly Leu Pro Asp Ser Leu Pro 275 280 285 Gly Asn Thr Asp Ile Thr Glu Leu Val Ala Leu Met Arg His Asp Lys 290 295 300 Lys Ala Thr Ser Gly Leu Thr Phe Val Leu Asp Gly Pro Ser Gly Val 305 310 315 320 Glu Leu Val Ser Gly Ile Pro Glu Asp Val Val Leu Arg Thr Leu Ala 325 330 335 Ala Met Pro Arg Gly Thr Ala 340 113 164 PRT Streptomyces lavendulae 113 Met Glu Arg Val Glu Leu Ile Arg Trp Pro Val Glu Ser Glu Arg Arg 1 5 10 15 Glu Arg Cys Arg Asp Arg Gly Val Met Arg Ile Leu Val Leu Glu Ala 20 25 30 Gly Ala Glu Ala Pro Leu Cys Val Asp Pro Lys Glu Asp Trp Val Arg 35 40 45 Ala Pro Val Ser Thr Asp Asp Leu Arg Ala Arg Val Glu Ala Leu Arg 50 55 60 Leu Arg Gly Ala Ala Ala Glu Ser Arg Pro Glu Val Asp Pro Asn Gly 65 70 75 80 Val Leu Arg Phe Arg Trp Arg Ser Ala Leu Leu Ser Pro Thr Glu Ala 85 90 95 Arg Leu Val Ala Arg Leu Ala Glu Ser Tyr Ala Glu Val Val Ala Arg 100 105 110 Asp Asp Leu Leu Arg Pro Pro Pro Gly Arg Thr Val Pro Ser Arg Asn 115 120 125 Ala Leu Asp Leu His Ile Met Arg Ile Arg Arg Arg Leu Ala Ala Leu 130 135 140 Gly Leu Arg Val Arg Thr Val Arg Gly Arg Gly Tyr Val Leu Glu Ser 145 150 155 160 Ala Glu Gly Val 114 514 PRT Streptomyces lavendulae 114 Met Leu Asp Arg Arg Ser Val Ile Arg Val Gly Ala Gly Val Ala Ala 1 5 10 15 Ala Ala Ala Val Ala Gly Thr Ala Ala Thr Gly Ala Ala Ala Val Gly 20 25 30 Leu Pro Gly Val Arg Gly Arg Ala Ala Ser Arg Gly Val Asp Trp Ala 35 40 45 Ser Leu Arg Arg His Leu Ser Gly Glu Leu Val Leu Pro Ala Asp Thr 50 55 60 Gly Tyr Glu Arg Ala Arg Lys Leu Tyr Ser Gly Gln Phe Asp Gly Ile 65 70 75 80 Arg Pro Gln Ala Val Ala Tyr Cys Arg Thr Glu Glu Asp Val Arg Thr 85 90 95 Thr Leu Ala Phe Ala Gln Asp His Ala Leu Pro Leu Thr Pro Arg Ser 100 105 110 Gly Gly His Ser Phe Gly Gly Tyr Ser Thr Thr Asp Gly Ile Val Leu 115 120 125 Asp Val Ser Gly Phe His Ala Val Ser Leu Thr Arg Asn Thr Val Val 130 135 140 Met Gly Ala Gly Thr Gln Gln Val Asp Ala Leu Thr Ala Leu Ser Pro 145 150 155 160 Arg Gly Val Ala Val Ala Ser Gly Asn Cys Ala Gly Val Cys Pro Gly 165 170 175 Gly Phe Val Gln Gly Gly Gly Leu Gly Trp Gln Ser Arg Lys Phe Gly 180 185 190 Met Ala Cys Asp Arg Leu Val Ser Ala Arg Val Val Leu Ala Asp Gly 195 200 205 Arg Ala Val Thr Ala Ser Ala Thr Glu His Pro Asp Leu Phe Trp Ala 210 215 220 Met Arg Gly Gly Gly Gly Gly Asn Phe Gly Val Val Thr Gly Phe Glu 225 230 235 240 Leu Arg Pro Thr Asp Val Pro Ser Val Val Ser Tyr Asn Leu Thr Trp 245 250 255 Pro Trp Glu Ser Ala Arg Arg Val Ile Glu Ala Trp Gln His Trp Ile 260 265 270 Ile Asp Gly Pro Arg Asp Leu Gly Ala Ala Met Ala Val Gln Trp Pro 275 280 285 Asp Ala Gly Thr Gly Thr Pro Val Val Val Val Thr Gly Ala Trp Leu 290 295 300 Gly Ala Ala Asp Ala Leu Thr Pro Val Leu Asp Ser Leu Val Ala Ser 305 310 315 320 Val Gly Ser Ala Pro Ala Thr Arg Ser Ala Lys Ala Leu Ser Gln His 325 330 335 Asp Ala Met Met Ala Gln Tyr Gly Cys Ala Asp Leu Thr Pro Glu Gln 340 345 350 Cys His Thr Val Gly Tyr Ser Pro Glu Ala Ala Leu Pro Arg Gln Asn 355 360 365 Phe Ser Met Asp Arg Asn Arg Leu Phe Ser Arg Ala Ile Gly Gln Gly 370 375 380 Gly Val Glu Arg Ile Leu Glu Ala Phe Ala Ala Asp Pro Arg Ala Gly 385 390 395 400 Gln Phe Arg Phe Leu Ser Phe Phe Ala Leu Gly Gly Ala Ala Asn Arg 405 410 415 Pro Asp Arg Thr Thr Thr Ala Tyr Val His Arg Asp Thr Glu Phe Tyr 420 425 430 Leu Gly Phe Ser Ile Gly Leu Asn Asp Pro Glu Tyr Thr Ala Glu Asp 435 440 445 Glu Arg Leu Gly Arg Ala Trp Ala Ala Arg Gly Leu Arg Thr Leu Asp 450 455 460 Pro His Ser Asn Gly Glu Ser Tyr Gln Asn Phe Ile Asp Pro Glu Leu 465 470 475 480 Asp Asp Trp Lys Ser Ala Tyr Tyr Ala Glu Asn Tyr Val Arg Leu Ala 485 490 495 Ala Val Lys Ala Ala Tyr Asp Pro His Arg Leu Phe Ser Phe Ala Gln 500 505 510 Ala Val 115 315 PRT Streptomyces lavendulae 115 Met Thr Gly Asp Thr Asp Gly Ala Gly Gly Gly Asp Val Thr Phe Arg 1 5 10 15 Trp Pro Ala Ala Gly Asp Val Thr Ala Asp Leu Asp Leu Leu Ala Ala 20 25 30 Arg Val Arg Gly Leu Leu Gly His Arg Glu Asp Pro Leu Ala Gly Val 35 40 45 Gly Val Ala Met Pro Ala Ile Cys Asp Ala Ala Gly Thr Val Arg Thr 50 55 60 Trp Pro Gly Arg Pro Ser Trp Ala Gly Leu Asn Leu Thr Ala Ala Phe 65 70 75 80 Gly Gln Leu Leu Pro Gly Thr Pro Val Ala Cys Ala Asp Asp Gly Asp 85 90 95 Leu Ala Ala Leu Ala Glu Ser Arg Ala Ala Gly Cys Arg His Leu Leu 100 105 110 Tyr Val Gly Val Gly Thr Gly Ile Gly Gly Gly Ile Val His Glu Gly 115 120 125 Arg Ala Trp Pro Gly Pro Gly Arg Gly Ser Cys Glu Val Gly His Val 130 135 140 Val Val Asp Arg Ser Gly Pro Arg Cys Asp Cys Gly Arg Ala Gly Cys 145 150 155 160 Val Gln Ala Val Ala Ser Gly Pro Ala Thr Leu Arg Arg Ala Ala Glu 165 170 175 Arg Arg Gly Arg Glu Thr Gly Phe Asp Glu Leu Ala Ser Gly Ala Arg 180 185 190 Leu His Ala Pro Trp Ala Glu Ala Ala Val Asp Glu Ser Ala Ala Ala 195 200 205 Leu Ala Thr Ala Val Thr Gly Ile Cys Glu Leu Ala His Pro Glu Leu 210 215 220 Val Leu Val Gly Gly Gly Phe Ala Ala Gly Val Pro Gly Tyr Val Ala 225 230 235 240 Ser Val Ala Ala His Val Glu Arg Leu Thr Arg Pro Gly Thr Asp Pro 245 250 255 Val Arg Val Arg Pro Ala Val Leu Gly Gly Arg Ser Ser Leu His Gly 260 265 270 Ala Leu Leu Leu Ala Arg Glu Ala His Gly Arg Gly Asn Arg Pro Pro 275 280 285 Glu Ser Asp Arg Val Ser Ser Asp Val Ser Ser Asp Val Ser Phe Gly 290 295 300 Gly Val Thr Asp Arg Ala Val Gly Arg Ser Asp 305 310 315 116 514 PRT Streptomyces lavendulae 116 Met Pro Pro Ser Pro Arg Ala Leu Val Ile Gly Ile Asp Gly Gly Thr 1 5 10 15 Phe Asp Thr Val Asp Pro Leu Ile Glu Cys Gly Leu Leu Pro His Met 20 25 30 Ala Lys Leu Leu Arg Glu Ser Ala Ser Ala Ala Thr Asp Cys Thr Trp 35 40 45 Pro Ala His Thr Ala Pro Gly Trp Ser Thr Phe Val Ser Ala Ser Asp 50 55 60 Pro Gly Gly His Gly Ile Tyr Gln Phe Tyr Asp Thr Gln Asp Pro Ala 65 70 75 80 Tyr Gly Ala Arg Val Thr Arg Ser Gly Asp Leu Gly Arg Ser Cys Ala 85 90 95 Trp Asp Trp Leu Ala Ala Gln Glu Tyr Ser Leu Gly Leu Ile Asn Ile 100 105 110 Pro Met Ser His Pro Pro Ala Asp Leu Pro Gly Tyr Gln Val Thr Trp 115 120 125 Pro Leu Glu Arg Thr Leu Lys His Cys Arg Pro Asp Ser Leu Leu Arg 130 135 140 Glu Leu Ala Ala Ala Lys Ala His Phe Gln Ser Asp Leu Ala Thr Met 145 150 155 160 Phe Arg Gly Asp Met Ala Tyr Leu Glu Glu Ala Glu Arg Asn Val Ala 165 170 175 Ala Arg Val Arg Ser Val Arg His Leu Met Ser Thr Arg Pro Thr Asp 180 185 190 Val Val Met Val Val Leu Thr Glu Ala Asp Arg Val Gly His His Tyr 195 200 205 Trp His Tyr Gly Asp Pro Gly His Pro Gly His Arg Pro Ala Pro Glu 210 215 220 Gly Ser Gly Trp Asp Val Ala Met Pro Arg Ile Tyr Gln Ala Ile Asp 225 230 235 240 His Ala Val Gly Glu Leu Leu Glu Leu Val Asp Glu Asp Thr Ser Val 245 250 255 Val Leu Val Ser Asp His Gly Leu Gly Thr Gly Arg His Gly Leu Ser 260 265 270 Val His Thr Leu Leu Glu Glu Ala Gly Leu Leu Ala Thr Ala Pro Gly 275 280 285 Glu Glu Pro Gln Asp Ala Ala Ala Ser Trp Phe Ala Gly Asn Gly Arg 290 295 300 His Val Asp Phe Arg Arg Thr Ser Val Tyr Met Pro Val Pro Gly Ser 305 310 315 320 Tyr Gly Leu Asn Ile Asn Val Arg Gly Arg Gln Gln Arg Gly Thr Val 325 330 335 Ala Pro Arg Asp Arg Glu Arg Val Met Asp Glu Val Thr Gly Leu Leu 340 345 350 Ser Gly Leu Thr Gly Pro Glu Gly Gln Gln Val Phe Arg Ala Val Arg 355 360 365 Pro Arg Glu Glu Ala Tyr Pro Gly Pro His Thr Gly Arg Ala Pro Asp 370 375 380 Leu Leu Leu Val Pro Arg Asp Glu Thr Val Leu Pro Val Pro Asp Leu 385 390 395 400 Gly Gly Asp Val Trp Arg Pro Ser Ala Gln Thr Gly Leu His Arg Tyr 405 410 415 Arg Gly Leu Trp Ala His Arg Ser Pro Arg Val Arg Pro Gly Arg Leu 420 425 430 Pro Gly Thr Val Ala Leu Thr Asp Thr Leu Pro Thr Leu Leu Thr Asp 435 440 445 Leu Gly Ala Ala Trp Pro Ser Asp Ile His Gly Arg Pro Val Thr Ala 450 455 460 Val Leu Asp Asp Gly Val Arg Val Pro Pro Ser Asp Pro Arg Val Glu 465 470 475 480 Ala Thr Gly Thr Pro Ala Thr Thr Ile Pro Ala Ala Ala Ser Ala Ala 485 490 495 Asp Ala Ala Glu Asp Ala Tyr Thr Ser Asp Arg Leu Arg Glu Met Gly 500 505 510 Tyr Leu 117 93 PRT Streptomyces lavendulae 117 Met Glu Thr Leu Thr Thr Asp Lys Ile Lys Asp Arg Leu Arg Lys Val 1 5 10 15 Leu Val Asp Ser Leu Glu Leu Ser Leu Asp Pro Ser Ala Val Pro Asp 20 25 30 Glu Gly Leu Val Glu Lys Leu Gly Leu Asp Ser Ile Asn Thr Ile Glu 35 40 45 Phe Leu Ile Trp Val Glu Ser Glu Phe Gly Ile Glu Ile Ala Asp Glu 50 55 60 Asp Leu Ser Ile Lys Leu Ile Asp Ser Leu Asp Leu Leu Ala Gly Tyr 65 70 75 80 Val Ser Glu Arg Val Asn Gly Val Thr Ala Pro Ala Glu 85 90 118 470 PRT Streptomyces lavendulae 118 Met Asp Arg His Ala Leu Val Ile Gly Leu Asp Gly Met Pro Arg Thr 1 5 10 15 Leu Leu Thr Arg Leu Ala Gly Asp Gly Thr Met Pro His Thr Ala Ala 20 25 30 Leu Leu Ala Glu Gly His Cys Ala Glu Leu Leu Ala Pro Val Pro Glu 35 40 45 Ile Ser Ser Thr Ser Trp Ala Thr Phe Leu Thr Gly Thr Asn Pro Gly 50 55 60 Arg His Gly Ile Tyr Gly Phe Thr Asp Leu Ala Pro Gly Asp Gly Tyr 65 70 75 80 Arg Ile Thr Phe Pro Gly Val Arg Gln Leu Arg Glu Pro Pro Leu Trp 85 90 95 Glu Leu Ala Ala Arg Ala Gly Arg Arg Thr Val Cys Leu Asn Val Pro 100 105 110 Gly Thr Tyr Pro Ala Pro Ala Ile Asp Gly Val Leu Val Ser Gly Phe 115 120 125 Val Ala Pro Glu Leu Glu Arg Ala Val Ser Pro Pro Arg Leu Leu Pro 130 135 140 Leu Leu Arg Gly Leu Asp Tyr Glu Leu Asp Val Glu Val Gly Asp Val 145 150 155 160 Ala Ala Asp Pro Ala Ala Phe Leu Gly Arg Ala Val Arg Ala Leu Arg 165 170 175 Ala Arg Thr Arg Ala Met Glu His Leu Leu Arg Gln Glu Thr Trp Asp 180 185 190 Leu Ala Val Ala Val Leu Thr Glu Thr Asp Arg Val His His Phe Leu 195 200 205 Trp Arg Ala Val Ala Asp Pro Ala Asp Pro Leu His Gly Asp Val Leu 210 215 220 Ala Phe Tyr Arg Leu Val Asp Asp Cys Val Ala Thr Leu Val Ser Thr 225 230 235 240 Leu Pro Pro Gly Gly Glu Leu Phe Leu Met Ser Asp His Gly Phe Gly 245 250 255 Pro Ala Ala Cys Gln Val Tyr Leu Asn Ala Trp Leu Arg Glu Ser Gly 260 265 270 Trp Leu Ala Gly Leu Asp Val Cys Pro Asp Leu Thr Ala Val Asp Ala 275 280 285 Arg Ser Thr Ala Phe Ala Leu Asp Pro Ala Arg Ile His Leu Asn Arg 290 295 300 Lys Ser Arg Phe Pro Gly Gly Gly Leu Thr Asp Ala Glu Ala Asp Glu 305 310 315 320 Ala Ala His Glu Ile Ala Arg Glu Leu Ser Ala Leu Arg Cys Asp Gly 325 330 335 Thr Arg Leu Gly Pro Asp Val Asp Gly Pro Leu Leu Val Arg Asp Leu 340 345 350 Tyr Arg Ala Gln Glu Ile Tyr His Gly Pro Leu Leu Gly Asn Ala Pro 355 360 365 Asp Leu Val Ala Val Pro Ala Pro Gly Val Gln Leu Arg Gly Gly Trp 370 375 380 Gly Gly Thr His Thr Val Arg Asn Asp Ile Leu Thr Gly Thr His Thr 385 390 395 400 Arg Asp Asp Ala Val Phe Tyr Arg Arg Gly Ala Pro Ala Pro Ala Pro 405 410 415 Gly Ala Asp Asp Gly Pro Leu Asp Met Thr Asp Val Ala Pro Thr Val 420 425 430 Leu Ala Ser Leu Gly Ile His Pro Gly Gly Leu Asp Gly Ala Ala Val 435 440 445 Leu Gly Thr Thr Gly Pro Ala Ser Gly His Gly Arg Thr Asp Pro Pro 450 455 460 Leu Asp Ile Arg Glu Leu 465 470 119 611 PRT Streptomyces lavendulae 119 Met Lys His Asp Leu Gly Leu Ala Pro Ser Ala Pro Lys Pro Gly Thr 1 5 10 15 Leu Asp Leu Ser Leu Asp Pro Arg Ile Thr Asp Pro Ala Ser Phe Arg 20 25 30 Val Ser Phe Leu Ile Leu Leu Asp Gly Asp Leu Val Met Ser Pro Glu 35 40 45 His Leu Gly Val Ala Tyr Met Ala Gly Val Leu Arg His Thr Gly Phe 50 55 60 Thr Ala Glu Ile Arg Glu Val Glu His Gly Asp Asp Gln Ala Ala Ala 65 70 75 80 Thr Val Glu Ala Leu Lys Glu Tyr Arg Pro Asp Leu Val Cys Phe Thr 85 90 95 Leu Met Ser Leu Asn Leu Gly Ser Cys Leu Thr Leu Cys Arg Met Leu 100 105 110 Arg Glu Glu Leu Pro Gly Thr Thr Ile Ala Cys Gly Gly Pro Ala Gly 115 120 125 Thr Phe Ala Gly Leu Asp Val Leu Arg Asn Asn Pro Trp Thr Asp Val 130 135 140 Val Ala Val Gly Glu Gly Glu Pro Thr Ile Leu Asp Leu Val Gln Arg 145 150 155 160 Leu Tyr Leu Lys Glu Pro Leu Ser Ala Cys Lys Gly Ile Cys Tyr Arg 165 170 175 Asp Glu Asp Gly Thr Pro Arg Gln Asn Pro Ala Arg Pro Leu Ile His 180 185 190 Asn Leu Glu Asp Leu Pro Phe Pro Ala Arg Asp Gln Leu Arg Gln His 195 200 205 Gly Asp Lys Leu Glu Tyr Val Arg Val Ser Thr Ser Arg Gly Cys Val 210 215 220 Ala Asn Cys Ala Phe Cys Ser Ala Pro His Leu Lys Asn Arg Val Gln 225 230 235 240 Ala Gly Lys Ala Trp Arg Gly Arg Gly Pro Glu Gln Ile Val Asp Glu 245 250 255 Val Ala Glu Ile Val Glu Arg His Gln Phe Arg Thr Phe Asp Phe Val 260 265 270 Asp Ser Thr Phe Glu Asp Pro Asp Gly Gly Arg Val Gly Lys Lys Arg 275 280 285 Val Ala Ala Ile Ala Asn Gly Ile Leu Glu Arg Gly Leu Asp Ile Tyr 290 295 300 Tyr Asn Val Cys Met Arg Ala Glu Asn Trp His Asp Thr Pro Glu Asp 305 310 315 320 His Ala Leu Leu Asp Leu Leu Val Ala Ser Gly Leu Glu Lys Val Asn 325 330 335 Val Gly Ile Glu Ala Gly Thr Ala Glu Glu Leu Leu Leu Trp Glu Lys 340 345 350 Arg Ala Thr Val Glu Asp Asn Val Thr Ile Ile Arg Met Leu Arg Glu 355 360 365 His Gly Ile Tyr Leu Ala Met Gly Phe Ile Pro Phe His Pro Tyr Ala 370 375 380 Thr Leu Glu Thr Ile Val Thr Asn Ala Ala Phe Leu Arg Asp Asn Ser 385 390 395 400 Gly His Asn Leu Arg Arg Met Thr Glu Arg Leu Glu Ile Tyr Pro Gly 405 410 415 Thr Ala Ile Val Ser Arg Met Arg Ala Asp Gly Leu Leu Gly Glu Ser 420 425 430 Tyr Leu Glu Gly Leu Asp Pro Tyr Gly Tyr Ala Phe Lys Asp Pro Arg 435 440 445 Val Gly Arg Leu Ala Lys His Phe Ala Gln Leu Tyr Asn Asn Asp Asp 450 455 460 Tyr His Arg His Gly Val Ile Thr Glu Gln Ser Ser Val Phe Ala Phe 465 470 475 480 Glu Thr Tyr Asn Val Val Leu Gln Thr Phe Ile Ser Arg Leu His Arg 485 490 495 Arg Phe Thr Thr Leu Pro Gly Val Asp Glu Val Met Glu Ala Phe Lys 500 505 510 Ala Arg Val His Glu Ile Arg Gln Glu Met Gly Arg His Asn Tyr Gly 515 520 525 Phe Phe Met Ser Asn Val Glu Ala Val Met Asn Asp Thr Leu Asp Pro 530 535 540 Glu Lys Gln Arg Arg Gln Val Val Asp Val Glu His Phe Phe Arg Asp 545 550 555 560 Arg Leu Asp Val Leu Arg Ser Glu Gln Leu Arg Val Gly Lys Ala Leu 565 570 575 Thr Arg Leu Gly Ala Arg Val Thr Glu Val Ser Ser Thr Ile Pro Lys 580 585 590 Glu Arg Pro Gly Gly Leu Pro Arg Gln Tyr Thr Gly Glu Gly Ser Gly 595 600 605 Ala Thr Trp 610 120 359 PRT Streptomyces lavendulae 120 Met Pro Arg Gly Glu Thr Gly Thr Ala Ala Ala Arg Val Ala Val Cys 1 5 10 15 Thr Leu Ser Ser Arg Glu Leu Val Gly Pro Leu Ala Arg Leu Pro Gly 20 25 30 Val Ala Ala Ala Gly Thr Leu Met Thr Ala Asn Leu Gly Ile Glu Gln 35 40 45 Val Ile Lys Ala Leu Arg Cys Asp Arg Thr Val Arg Gly Leu Leu Val 50 55 60 Cys Gly Arg Asp Ser Pro Arg Phe Arg Ala Gly Gln Ser Leu Ile Ala 65 70 75 80 Leu Phe Arg His Gly Leu Arg Pro Glu Asp Gly His Ile Arg Gly Ala 85 90 95 Thr Gly Tyr Leu Pro Val Leu Arg Ser Val Thr Ala Arg Glu Thr Glu 100 105 110 Glu Val Arg Ala Arg Val Glu Leu Val Asp Ala Arg Gly Glu Arg Asp 115 120 125 Val Glu Thr Leu Arg Ala Glu Val Ala Ala Leu Leu Ala Arg Val Arg 130 135 140 Arg Thr Pro Ala Leu Pro Ser Arg Glu His Asp Gly Gly Gln Pro Ser 145 150 155 160 Phe Val Glu Pro Asp Phe Gly Arg Leu His Pro Val Gly Arg Arg Arg 165 170 175 Ser Leu Asp Ala Gly Ile Gly Gly Phe Val Leu Ile Ser Val Asp Arg 180 185 190 Glu His Arg Arg Ile Leu Leu Arg His Tyr Thr Ser Asp Val Arg Pro 195 200 205 Arg His Glu Met Trp Gly Thr Arg Gly Glu Ala Met Leu Leu Gly Leu 210 215 220 Leu Glu Ala Gly Val Ile Glu Asp Pro Ala His Ala Gly Tyr Leu Gly 225 230 235 240 Ala Glu Leu Ala Lys Ala Glu Thr Ala Leu Arg Leu Gly Leu His Tyr 245 250 255 Glu Gln Asp Leu Pro Leu Arg Pro Pro Gly Arg Pro Pro Gly Pro Val 260 265 270 Arg Arg Arg Thr Ala Lys Glu Arg Thr Thr Met Ala Gln Ala Pro Ala 275 280 285 Leu Glu Asp Phe Leu Arg Leu Val Thr Arg Thr Leu Gly Ala Glu Asp 290 295 300 Ala Val Leu Asp Leu His Thr Pro Leu Gly Glu Gln Leu Ala Val Asp 305 310 315 320 Ser Ala Arg Leu Ile Glu Leu Thr Val Val Leu Glu Glu Glu Leu Gly 325 330 335 Ala Asp Leu Pro Asp Asp Ala Asp Leu Ala Arg Ala Thr Pro Ala Glu 340 345 350 Leu His Lys Ala Leu Val Gly 355 121 145 PRT Streptomyces lavendulae 121 Met Arg Ser Val Leu Leu Leu Asn Gly Pro Asn Leu Gly Thr Leu Gly 1 5 10 15 Lys Arg Gln Pro Glu Ile Tyr Gly Thr Asp Thr Leu Ala Glu Ile Glu 20 25 30 Ala Ala Val Ala Glu Glu Val Gly Ala Arg Gly Trp Glu Val Val Ser 35 40 45 Glu Gln Arg Asn Gly Glu Gly Glu Leu Val Asp Val Leu Gln Arg His 50 55 60 Asp Asp Val Val Gly Ala Val Val Asn Pro Gly Ala Leu Met Ile Ala 65 70 75 80 Gly Trp Ser Leu Arg Asp Ala Leu Ala Asp Phe Ala Pro Pro Trp Val 85 90 95 Glu Val His Leu Ser Asn Val Trp Gly Arg Glu Ala Phe Arg His Thr 100 105 110 Ser Val Thr Ala Pro Leu Ala Ser Gly Val Val Met Gly Met Gly Ala 115 120 125 Leu Gly Tyr Arg Leu Ala Ala Arg Ala Leu Thr Arg Leu Val Pro Glu 130 135 140 Asp 145 122 177 PRT Streptomyces lavendulae 122 Met Gly Arg Tyr Gly Arg Glu Gly Leu Gly Met Ser Arg Thr Ala Glu 1 5 10 15 Gly Asn Ala Gly Gly Val Val Val Pro Val Val Arg Leu Val Ala Val 20 25 30 Thr Asp Gly Pro Asp Ala Glu Gly Trp Arg Gln Ala Leu Ala Pro Glu 35 40 45 Leu Val Val Glu His Gly Val Glu Ala Ile Ala Glu Ala Ala Gly Asp 50 55 60 Gly Gly Pro Trp Ala Leu Val Cys Ala Gly Ala Gly Leu Gly Ala Ala 65 70 75 80 Leu Arg Ala Ala Glu Arg Ala Ala Arg Pro Pro Val His Val Leu Leu 85 90 95 Trp Leu Gly Ser Arg Gly Pro Gly Glu Gly Val Gly Gly Glu Val Ser 100 105 110 Gly Gln Phe Pro Cys Pro Val Thr Ala Leu Val Ser Ala Glu Val Asp 115 120 125 Arg Gly Arg Ala Val Val Pro Ala Trp Arg Gly Leu Thr Glu Gly Pro 130 135 140 Phe Thr Val Arg Ile Leu Pro Ala Ala Cys Pro Leu Pro Gly Ala Cys 145 150 155 160 Asp Gln Ala Gly Ala Gln Val Ile Lys Glu Glu Leu Arg Val Trp Pro 165 170 175 Ala 123 254 PRT Streptomyces lavendulae 123 Met Asp Ala Thr Leu Thr Asn Asp Val Glu Lys Ala Ser Arg Asp Leu 1 5 10 15 Val Glu Ala Gly Tyr Cys Leu Ile Glu Cys Pro Leu Pro Ala Ala Val 20 25 30 Phe Glu Lys Leu Arg Gly Arg Leu Leu Glu Val Ala Glu Gln Glu Arg 35 40 45 Glu Asn Gly Ser Ala Phe Leu Tyr Asp Gly Gly Asn Gln Arg Val Phe 50 55 60 Ser Leu Leu Asn Lys Gly Glu Glu Phe Glu Gln Asn Val Gln Asp Pro 65 70 75 80 Thr Val Met Leu Leu Met Glu Glu Ile Leu Gly Phe Gly Phe Leu Leu 85 90 95 Ser Ser Thr His Ala Asn Ile Ala Gly Pro Gly Gly Ser Arg Met His 100 105 110 Leu His Ala Asp Gln Thr Phe Ala Arg Pro Pro Trp Pro Pro Tyr Pro 115 120 125 Leu Val Ala Asn Ser Met Trp Met Leu Asp Asp Phe Thr Glu Asp Asn 130 135 140 Gly Ala Thr Arg Leu Val Pro Gly Ser His Leu Leu Gly Arg Gln Pro 145 150 155 160 Asp Tyr Asp Arg Gly Glu Gly Asn Thr Glu Thr Val Ala Val Cys Ala 165 170 175 Pro Ala Gly Ser Val Met Val Phe Asp Gly Arg Leu Trp His Gln Thr 180 185 190 Gly Ala Asn Thr Thr Asp Arg Pro Arg His Gly Ile Leu Asn Tyr Tyr 195 200 205 Cys Arg Gly Tyr Val Arg Gln Gln Gln Asn Phe Phe Ser Gly Leu Arg 210 215 220 Glu Asp Val Ala Thr Arg Ala Thr Pro Glu Leu Arg Arg Leu Leu Gly 225 230 235 240 Tyr Glu Asn Tyr Phe Ser Leu Gly Met Thr Asp Gly Leu Pro 245 250 124 264 PRT Streptomyces lavendulae 124 Met Ala His Ser Pro Arg Arg Pro Asp Gly Pro Leu Arg Ile Gly Val 1 5 10 15 Trp Leu Ala Pro Gln His Thr Ser Val Ala Glu Leu Arg Ala Ala Trp 20 25 30 Arg Ala Ala Asp Ser Leu Gly Val Asp Ser Leu Trp Leu Trp Asp His 35 40 45 Phe Phe Pro Leu Thr Gly Asp Pro Asp Gly Ser His Phe Glu Ala Trp 50 55 60 Thr Leu Leu Ala Ala Met Ala Ala Asp Thr Arg Ala Ala Arg Leu Gly 65 70 75 80 Thr Leu Val Ser Asn Tyr Ala Tyr Arg Asn Pro Asp Leu Leu Ala Asp 85 90 95 Met Ala Arg Thr Val Asp His Ile Gly Asp Gly Arg Leu Ile Leu Gly 100 105 110 Met Gly Ala Gly Trp Val Glu Arg Asp Leu Lys Glu Tyr Gly Tyr Pro 115 120 125 Thr Pro Gly Ala Gly Glu Arg Val Asp Gly Leu Ile Glu Ala Val Glu 130 135 140 Arg Val Asp Arg Arg Leu Gly Arg Leu Arg Pro Gly Pro Leu Gly Asp 145 150 155 160 Leu Pro Leu Leu Ile Gly Gly Asp Gly Gln Arg Arg Leu Leu Arg Phe 165 170 175 Ala Ala Glu Arg Ala Ala Ile Trp Asn Thr Met Ala Trp Arg Phe Ala 180 185 190 Glu Gly Asn Arg Val Leu Asp Glu Trp Cys Ala Arg Val Gly Arg Asp 195 200 205 Pro Ala Glu Ile Glu Arg Ser Ala Phe Val Thr Arg Asp Gln Thr Asp 210 215 220 Glu Glu Leu Arg Cys Leu Val Ala Thr Gly Val Gln His Leu Ile Phe 225 230 235 240 Gln Val Gly His Pro Phe Arg Phe Asp Gly Val Glu Arg Ala Leu Arg 245 250 255 Phe Ala Gly Gly Trp Ser Lys Gly 260 125 274 PRT Streptomyces lavendulae 125 Met Lys Ile Ser Ile Ala Leu Pro Asn Thr Val Pro Gly Ala Asp Gly 1 5 10 15 Arg Leu Ile Thr Asp Trp Ala Arg Arg Ala Glu Glu Arg Gly Phe Ala 20 25 30 Ser Leu Ala Ala Thr Glu Arg Leu Val Tyr Pro Gly His Asp Pro Leu 35 40 45 Leu Ala Leu Ala Ala Ala Ala Gly Ala Thr Ser Arg Ile Gly Leu Leu 50 55 60 Thr Asn Val Leu Ile Gly Pro Leu Arg Thr Ala Pro Val Leu Ala Lys 65 70 75 80 Ala Val Ala Ser Leu Asp Ser Leu Ser Gly Gly Arg Phe Thr Leu Gly 85 90 95 Val Gly Pro Gly Val Arg Glu Asp Asp Phe Glu Ala Ala Gly Arg Ala 100 105 110 Phe Asp Asp Arg Arg Ala Ala Phe Glu Glu Gln Leu Glu Leu Leu Gly 115 120 125 Arg Gly Ala Arg Pro Gly Ala Glu Gly Pro Gly Val Pro Val Leu Val 130 135 140 Gly Gly Val Ser Ala Ala Ala Val Arg Arg Val Ala Arg Trp Ala Asp 145 150 155 160 Gly Trp Thr Ala Pro Gly Leu Glu Pro Glu Arg Ile Val Pro Val Ala 165 170 175 Glu Arg Val Arg Arg Ala Trp Ser Glu Ala Gly Arg Ala Gly Ala Pro 180 185 190 His Val Val Ala Leu Ala Arg Tyr Thr Leu Gly Glu Asp Val Ala Gln 195 200 205 Glu Ser Ala Ala Phe Val Arg Asp Tyr Phe Ala Val Leu Gly Glu Glu 210 215 220 Ala Glu Glu Phe Val Ala Lys Thr Pro Arg Thr Ala Gly Gln Leu Arg 225 230 235 240 Ala Ala Val Ser Ala Leu Ala Asp Gly Gly Val Asp Glu Val Val Leu 245 250 255 His Pro Thr Ala Ala Ala Leu Ser Gln Val Asp Arg Leu Ala Asp Ala 260 265 270 Leu Leu 126 460 PRT Streptomyces lavendulae 126 Met Pro Ala Ala Gly Lys Val Ala Val Ile Gly Leu Asp Ser Ala Thr 1 5 10 15 Pro Gln Tyr Met Phe Asp Arg Phe Ala Glu Asp Met Pro Val Phe Thr 20 25 30 Ala Leu Arg Arg Lys Ser Leu Trp Gly Pro Met Arg Ser Ile Asp Pro 35 40 45 Pro Ile Thr Met Pro Ala Trp Ser Cys Met Met Ser Gly Arg Ser Pro 50 55 60 Gly Glu Leu Gly Val Tyr Gly Phe Arg Asp Arg Gly Ala Tyr Asp Tyr 65 70 75 80 Gly Pro Leu Lys Phe Ala Thr Ser His Ser Ile Gln Ala Pro Arg Ile 85 90 95 Trp Asp Glu Met Thr Ala Ala Gly Arg Ser Ser Val Val Leu Gly Val 100 105 110 Pro Gly Thr Tyr Pro Pro Ala Pro Ile Arg Gly Ala Met Val Ser Cys 115 120 125 Phe Leu Ala Pro Ser Thr Gln Ser Arg Tyr Thr Ser Pro Pro Gly Leu 130 135 140 Ala Asp Glu Leu Glu Lys Leu Thr Gly Gly Tyr Ala Leu Asp Val Glu 145 150 155 160 Asp Phe Arg Ser Thr Asp Leu Glu Arg Val Ser Gln Arg Val Phe Asp 165 170 175 Met Ser Glu Gln Arg Phe Glu Val Ala Arg His Leu Ala Thr Thr Gln 180 185 190 Glu Trp Asp Phe Leu Ser Phe Val Asp Met Gly Pro Asp Arg Leu His 195 200 205 His Gly Phe Trp Lys Tyr Cys Asp Pro Asp His Pro Arg His Glu Pro 210 215 220 Gly Asn Ala Tyr Ala Gly Leu Phe Arg Asp Tyr Tyr Arg Ala Leu Asp 225 230 235 240 Arg His Leu Gly Arg Phe Leu Glu Ser Leu Pro Glu Asn Thr Thr Val 245 250 255 Leu Val Val Ser Asp His Gly Ala Gln Pro Met Val Gly Gly Leu Phe 260 265 270 Val Asn Glu Trp Leu Arg Lys Glu Gly Tyr Leu Val Leu Thr Glu Glu 275 280 285 Pro Ala Gly Pro Thr Pro Val Ala Gln Ala Ala Val Asp Trp Lys Arg 290 295 300 Thr Thr Ala Trp Ala Glu Gly Gly Tyr Tyr Gly Arg Ile Phe Leu Asn 305 310 315 320 Val Glu Gly Arg Glu Pro Gln Gly Thr Ile Pro Ala Ala Glu Tyr Glu 325 330 335 Ser Thr Arg Asp Leu Ile Ala Ser Ala Leu Glu Ala Leu Pro Asp Asp 340 345 350 Gln Gly Gln Pro Met Gly Thr Arg Ala Leu Arg Pro Gly Glu Leu Tyr 355 360 365 Gly Glu Val Asn Gly Ile Ala Pro Asp Leu Leu Val Tyr Val Gly Asn 370 375 380 Leu Arg Trp Arg Ala Leu Ala Thr Leu Gly Met Gly Lys Gly Leu Tyr 385 390 395 400 Thr Thr Glu Asn Asp Thr Gly Pro Asp His Ala Asn His Gly Asp Thr 405 410 415 Gly Ile Phe Ala Leu Ser Ala Pro Gly Ile Thr Pro Gly Arg Ala Asp 420 425 430 Gly Leu Ser Leu Tyr Asp Val Ala Pro Thr Leu Arg Glu Leu Leu Gly 435 440 445 Leu Ala Pro Gln Gly Ser Arg Gly Ser Leu Leu Gly 450 455 460 127 511 PRT Streptomyces lavendulae 127 Met Lys Ala Met Asp Arg Val Asp Arg Ala Val Glu Arg Phe Pro Met 1 5 10 15 Tyr Ile Asp Gly Gln Ala Val Gln Ala His Asp Gly Ala Val Leu Arg 20 25 30 Thr Phe Glu Pro Ala Thr Arg Arg His Leu Ala Asp Leu Pro Ser Gly 35 40 45 Gly Ala Glu Asp Val Arg Arg Ala Val Ser Ala Ala Arg Arg Ala Phe 50 55 60 Asp Glu Gly Pro Trp Pro Arg Met Ala Pro Gly Glu Arg Ala Gly Leu 65 70 75 80 Leu Arg Lys Ala Ala Gln Arg Leu Arg Glu Glu Ala Glu Pro Leu Ala 85 90 95 Glu Leu Glu Ala Arg Asp Asn Gly Ser Thr Leu Arg Lys Ala Leu Gly 100 105 110 Ala Asp Val Pro Gly Ala Ala Ala Ala Phe Glu Trp Ser Ala Trp Trp 115 120 125 Ala Glu His Val Pro Glu Arg Gln Pro Glu Ala Pro Gly Ser Gly Ser 130 135 140 Tyr Val Val Trp Arg Pro Val Gly Val Val Ala Ala Ile Val Pro Trp 145 150 155 160 Asn Leu Pro Leu Leu Leu Ala Ala Trp Arg Ile Ala Pro Ala Ile Ala 165 170 175 Ala Gly Asn Thr Cys Val Ile Lys Pro Ala Ser Phe Ala Ser Leu Ser 180 185 190 Thr Leu Arg Leu Val Glu Leu Leu His Glu Cys Gly Leu Pro Pro Gly 195 200 205 Val Val Asn Val Val Thr Gly Pro Gly Gly Val Ala Gly Glu Gln Leu 210 215 220 Val Arg Ser Pro Gly Val Asp Leu Val Ala Phe Thr Gly Ser Asp Glu 225 230 235 240 Thr Gly Ala Ala Val Arg Glu Gly Ala Ala Ala Ala Gly Thr Ser Ala 245 250 255 Arg Leu Asp Leu Gly Gly Lys Ser Pro Asn Ile Val Leu Ala Asp Ala 260 265 270 Asp Leu Asp Arg Ala Val Thr Gly Val Thr Trp Gly Ala Phe Leu His 275 280 285 Asn Gly Gln Val Cys Met Ala Gly Thr Arg Ala Val Val His Ala Asp 290 295 300 Val His Asp Asp Phe Leu Arg Leu Leu Ser Glu Arg Val Gly Arg Leu 305 310 315 320 Arg Val Gly Asp Pro Leu Asp Pro Ala Thr Asp Leu Gly Pro Leu Val 325 330 335 Ser Arg Asn Gln Ala Arg Thr Ala Arg Arg Phe Thr Glu Leu Gly Leu 340 345 350 Ser Gln Gly Ala Glu Leu Val Cys Gly Gly Arg Ala Pro Ala Ala Asp 355 360 365 Glu Leu Pro Pro Gly Leu Asp Ala Gly Ala Tyr Phe Leu Pro Thr Val 370 375 380 Leu Ala Ser Val Gly Ala Asp Asp Ala Val Ala Gln Glu Glu Ile Phe 385 390 395 400 Gly Pro Val Leu Ala Val Val Arg Ala Gly Ser Asp Asp Asp Ala Val 405 410 415 Arg Ile Ala Asn Gly Ser Arg Tyr Arg Leu Ser Ala Gly Val Trp Ser 420 425 430 Ala Asp Pro Ala Arg Ala Arg Ala Val Ala Glu Arg Leu Arg Ala Asp 435 440 445 Arg Val Trp Ile Asn Asp Tyr Arg Leu Val Asp Leu Glu Leu Pro Gly 450 455 460 Thr Ala Gly Pro Arg Ser Ala Val Trp Asp Arg Leu Thr Asn Glu Leu 465 470 475 480 Asp Ala Tyr Arg His Lys His Val Val His Gly Gly Gly Ala Gly Ala 485 490 495 Gly Gly Val Pro Ala Pro Pro Thr Pro Tyr Ala Leu Leu Gly Gly 500 505 510 128 472 PRT Streptomyces lavendulae 128 Met Lys Pro Ala Ser His Ser Val Thr Asp Thr Ser Ala Ala Leu Gly 1 5 10 15 Ala Ala Ala Ala Glu Glu Leu Ala Ala Gln Val Ala Gly Ser Val Leu 20 25 30 Leu Pro Gly Asp Glu Gly Tyr Asp Glu Glu Arg Ser Gly Phe Glu Leu 35 40 45 Ser Val Glu His Arg Pro Ala Leu Val Val Val Ala Thr Gly Ala Ala 50 55 60 Asp Val Ile Ala Ala Val Arg Phe Ala Arg Ala Arg Gly Leu Gly Ile 65 70 75 80 Ala Val Gln Ala Thr Gly His Gly Lys Ser Ser Ala Ala Thr Asp Val 85 90 95 Leu Ile Ser Thr Arg Arg Met Thr Gly Val Arg Val Asp Pro Arg Ala 100 105 110 Arg Thr Ala Arg Ile Glu Ala Gly Val Arg Trp Glu Gln Val Ile His 115 120 125 Glu Ala Ala Ala His Gly Leu Ala Pro Leu Ser Gly Ser Ala Pro Phe 130 135 140 Val Gly Ala Val Ser Tyr Leu Leu Gly Gly Gly Leu Gly Leu Leu Ser 145 150 155 160 Arg Lys Tyr Gly Phe Ala Gly Asp His Val Val Ser Leu Asp Leu Val 165 170 175 Thr Ala Asp Gly Arg Phe Leu Gln Val Ser Ala Glu Glu His Pro Asp 180 185 190 Leu Phe Trp Gly Val Arg Gly Ser Arg Gly Asn Leu Gly Ile Val Thr 195 200 205 Ser Val Glu Val Gly Leu Phe Pro Val Thr Gln Val Tyr Gly Gly Gly 210 215 220 Leu Phe Phe Asp Ala Gly Ser Thr Arg Ala Val Leu Asn Thr Tyr Leu 225 230 235 240 Gln Trp Ala Pro Arg Met Pro Glu Asp Met Ala Ser Ser Val Phe Leu 245 250 255 Ala Ala Tyr Pro Asp Ala Glu Gly Val Pro Gly Pro Leu Arg Gly Arg 260 265 270 Phe Val Thr His Ile Arg Leu Ala Trp Leu Gly Asp Pro Glu Glu Gly 275 280 285 Glu Arg Arg Phe Ala Glu Leu Arg Ala Ala Gly Thr Val Val Met Asp 290 295 300 Thr Val Asp Thr Leu Pro Tyr Thr Arg Ala Gly Ile Ile His Asn Asp 305 310 315 320 Pro Pro Ala Pro Val Ser Ser His Ser Lys Thr Val Met Phe Gly Gln 325 330 335 Leu Asp Glu Ile Ala Val Asp Glu Ile Leu Arg Leu Ala Gly Pro Gly 340 345 350 Thr Asp Ala Leu Phe Gly Val Glu Leu Arg His Leu Gly Gly Ala Leu 355 360 365 Ala Arg Pro Pro Arg His Pro Ser Ala Val Gly His Phe Pro Glu Ala 370 375 380 Val Phe Asn Ala Tyr Val Gly Ser Leu Val Asp Pro Asp Thr Leu Ala 385 390 395 400 Ala Val Asp Ala Ala Gln Gln Glu Phe Val Asp Ser Met Arg Pro Trp 405 410 415 Thr Thr Pro Gly Val Cys Leu Asn Phe Leu Ala Gly His Asn Thr Ser 420 425 430 Arg Glu Thr Thr Arg Ser Ala Tyr Thr Pro Glu Asp Tyr Ala Arg Leu 435 440 445 Gln Ala Leu Lys Ser Gln Tyr Asp Pro Gly Asn Val Phe Arg Phe Asn 450 455 460 Pro Asn Ile Pro Pro Leu Pro Ala 465 470 129 395 PRT Streptomyces lavendulae 129 Met Thr Ser Ala Ala Pro Pro Ala Phe Pro Phe Pro Pro Gly Pro Gly 1 5 10 15 Gly Thr Val Pro Pro Glu Tyr Ala Arg Leu Leu Thr Asp Asp Pro Val 20 25 30 Ala Glu Val Arg Leu Ala Asp Gly Ser Arg Ile Trp Leu Val Thr Arg 35 40 45 His Glu Asp Val Arg Thr Val Leu Thr Asp Gly Arg Phe Ser Arg His 50 55 60 Arg Ala Ala Met Leu Pro Gly Ser Gly Phe Gly Arg Ser Gln Gly Ser 65 70 75 80 Gly Ile Val Asp Leu Asp Pro Pro Glu His Gly Arg Leu Arg Gly Pro 85 90 95 Val Val Ala Ala Phe Gly Ala Ser Arg Thr Ala Arg Phe Ala Pro Arg 100 105 110 Ile Glu Ala Ala Ala Glu Ala Ala Leu Asp Arg Leu Pro Ala Gly Ser 115 120 125 Gly Thr Val Asp Leu Val Ala Ala Tyr Thr Ala Pro Phe Ala Gly Arg 130 135 140 Val Thr Ala Glu Phe Leu Gly Leu Pro Gly Asp Arg Trp Gln Asp Val 145 150 155 160 Thr Ser Asp Val Glu Leu Leu Leu Leu Pro Arg Gly Ala Thr Glu Gln 165 170 175 Ala Leu Lys Glu Ala Arg Gly Arg Leu Gly Gln Val Leu Asp Glu Leu 180 185 190 Leu Ala Ala Arg Arg Ala Glu Pro Gly Asp Ser Val Thr Asp Thr Leu 195 200 205 Leu Asp Ala Glu Glu Leu Thr Asp Asp Asp Arg Arg Leu Leu Leu His 210 215 220 Gly Leu Ile Ile Ser Gly Phe Ile Thr Ile Arg Asp Leu Leu Ala Arg 225 230 235 240 His Leu Phe Gly Val Leu Ser Ser Pro Gly Leu Ala Ala Arg Leu Arg 245 250 255 Glu Asp Pro Ser Val Leu Pro Ser Ala Val Gln Glu Leu Leu Arg Tyr 260 265 270 Tyr Pro Ser Ser Asn Asp Gly Leu Leu Arg Val Ala Thr Glu Asp Val 275 280 285 Val Leu Ser Gly Arg Arg Val Ala Ala Gly Asp Ala Val Leu Pro Leu 290 295 300 Val Ser Ala Ala Ser Arg Asp Pro Glu Val Phe Ala Asp Pro His Val 305 310 315 320 Leu Asp Ile Glu Arg Val Ala Asp Arg Gly Ile Ala Phe Gly Ala Gly 325 330 335 Gln His Ala Cys Pro Ala Thr Gly Leu Ala Val Thr Glu Leu Thr Val 340 345 350 Gly Ile Gly Arg Leu Leu Ala Ala Phe Pro Arg Ile Ala Leu Ala Val 355 360 365 Pro Pro Glu Glu Val Glu His Ser Ser Glu Leu Leu Pro Leu Gly Val 370 375 380 Arg Ser Leu Pro Val Val Pro Gly Pro Arg Asn 385 390 395 130 474 PRT Streptomyces lavendulae 130 Met Leu Pro Glu Phe Gln Leu Gln Trp Asn Trp Leu Asp Ala Pro Ala 1 5 10 15 Gly Gly Gly Gly Glu Leu Gln Ala Thr Trp Ala Arg Leu Arg Ile Ala 20 25 30 Val Gly Ala Glu Thr Val Thr Leu Val Gln Glu Pro Gly Gln Gly Thr 35 40 45 Phe Arg Glu His Thr Thr Gly Ser Leu Tyr Pro Leu Ala Glu Trp Ile 50 55 60 Ala Phe Asn Trp Trp Ser Leu Val Ala Asp Ala Arg Pro Gly Thr Gln 65 70 75 80 Ile Ser Gln Leu Arg Phe Ala Tyr Arg His Gly Val Gly Asp Asn Arg 85 90 95 Gly Ser Trp Trp Met Arg Ser Arg Arg His Ile Leu Arg Ala Ala Cys 100 105 110 Asp Gly Phe Arg Trp Pro Asp Met Leu Phe Val Pro Glu Gly Arg Glu 115 120 125 Thr Arg Ile Val Trp Met Pro Asp Met Gly Pro Asp Val Arg Pro Gly 130 135 140 Asn Arg Phe Ala Ser Arg Gly Asn Ser Cys Val Glu Ser Ala Ala Phe 145 150 155 160 Thr Ala Thr Leu Ala Ser Phe Val Asp Ala Val Thr Glu Arg Leu Thr 165 170 175 Asp Gln Gly Ile Thr Gly Thr Pro Leu Gln Glu Glu Trp Ala Ala Val 180 185 190 Arg Ala Thr Asp Glu Asp Glu Ala Ala Phe Cys Arg Ile Ala Ala Arg 195 200 205 Leu Gly Leu Asp Pro Tyr Ala Glu Ala Glu Pro Tyr Glu Ala Asp Ile 210 215 220 Leu Lys Ala Ala Glu Gln Leu Ala Glu Pro Leu Ala Ser Asp Phe Phe 225 230 235 240 Asn Gly Val Arg Pro Glu Arg Ile Ala Asp Gln Leu Gln Trp Ile Ala 245 250 255 Arg Val Arg Thr Leu Met Gly Thr Ala Pro Ala Asp Thr Pro Leu Pro 260 265 270 Pro Ala Leu Val Glu Leu Arg Lys Asp Cys Ala Asp Leu Ser Glu Lys 275 280 285 Phe Phe Ala Pro Gly Arg Leu Asp Asn Pro Trp Asp Leu Gly Tyr Glu 290 295 300 Val Ala His Arg Val Arg Ala Trp Ala Gly Leu Asp Asp Thr Ala Pro 305 310 315 320 Phe Asp Pro Ala Pro Leu Met Gly Tyr Arg Thr Glu Gln Val Pro Tyr 325 330 335 Met Asp Arg Gly Leu Val Ala Leu Gly Thr Arg Arg Gly Ala Asp Gly 340 345 350 Pro Val Leu Val Ser Ser Arg Arg Phe Thr Asp Arg Pro Arg Arg Phe 355 360 365 Leu Gln Ala Arg Ala Leu Trp His Leu Ile Cys Asp Pro Asp Asp Thr 370 375 380 Phe Leu Ile Ala Ala Ala His Thr His Arg Gln His Val Ala Arg Gly 385 390 395 400 Phe Ala Leu Glu Val Leu Ala Pro Ala Lys Gly Val Ala Thr Leu Leu 405 410 415 Ala Asp Pro Gly His Leu Val Ser Ala Glu Asp Val Glu Val Ile Ala 420 425 430 Asp Asp Tyr Gly Cys Gly Asn Ile Val Val Glu His Gln Leu Asp Asn 435 440 445 Arg Val Leu Ala Lys Asp Phe Thr Trp Pro Gly His Ala Arg Ala Gly 450 455 460 Ala Pro Ala Gly Glu Arg Ser Arg Gly Ala 465 470 131 443 PRT Streptomyces lavendulae 131 Met Thr Ile Arg Gln Arg Val Val Val Val Ile Thr Glu Gly Ala Ala 1 5 10 15 Pro Glu Leu Leu Asp Arg Trp Cys Ala Gln Gly Leu Leu Pro Gly Phe 20 25 30 Ala Ala Leu Arg Ser Gln Gly Ala Ser Gly Pro Leu His Ala Glu Gly 35 40 45 Thr Pro Tyr Glu Pro Pro Gly Leu Leu Ser Val Leu Thr Gly Arg Arg 50 55 60 Ala Ala Asp His Gly Phe Tyr Ser Tyr Trp Thr Cys His Asp Pro Glu 65 70 75 80 Tyr Ala Pro Gln Val Leu Thr Pro Glu His Arg Arg His Pro Leu Leu 85 90 95 Trp Gln His Glu Val Phe Gln Gly Val Arg Phe Ala Ser Ile Gly Leu 100 105 110 Phe Gly Thr His Pro Pro Glu Pro Phe Asp Gly Ser Leu Ile Thr Tyr 115 120 125 Pro Met Tyr Ala Thr Leu His Ala Cys His Pro Arg Ser Leu Gln Arg 130 135 140 Thr Leu Ala Lys Lys Gly Ile Arg Pro Val His Asp Val Ser Ile Phe 145 150 155 160 Trp Thr Gly Gln Asp Arg Asp Glu Leu Leu Pro Ser Leu Leu Glu Ala 165 170 175 Asp Val Gln Arg Gly Arg Ala Ala Leu Ala Leu Leu Glu Glu Ser Asp 180 185 190 Val Val Ile Val Asn Leu Thr Ser Ile Asp Arg Cys Ser His Ile Tyr 195 200 205 Trp Gln Glu Leu Glu His Gly Pro Glu His Glu Arg Glu Ser Ala Val 210 215 220 Phe Ala Ala Tyr Arg Thr Cys Asp Gln Val Ile Gln Asp Ala Leu Arg 225 230 235 240 Ala Ala Asp Asp Arg Thr Ser Val Val Ala Phe Ser Glu Ile Gly Phe 245 250 255 Gly Pro Leu Arg Asn Tyr Cys Ser Ile Asn Asp Glu Met Glu Gln Ala 260 265 270 Gly Phe Leu Ala Thr Ala Glu Asp Gly Arg Val Glu Trp Ala Gly Ser 275 280 285 Ala Ala Phe Glu Ala Val Gln Gly Thr His Gly Val Asn Ile Asn Leu 290 295 300 Arg Asp Arg Tyr Lys His Gly Leu Val Pro Glu Arg Asp Tyr Glu Lys 305 310 315 320 Val Arg Thr Asp Val Ala Ala Ala Leu Leu Glu Arg Arg Asn Pro Arg 325 330 335 Thr Gly Arg Leu Phe Phe Asp Ala Val Arg Arg Arg Glu Glu Val Tyr 340 345 350 Pro Gly Glu Ala Thr Gln His Ala Pro Asp Leu Ile Leu Glu Pro Ala 355 360 365 Asp Trp Arg Tyr Leu Pro Leu Gly Asp Pro His Trp Ala Ser His Val 370 375 380 His Arg Asp Trp Gln Ser Gly Trp His Arg Arg Glu Ser Tyr Trp Ser 385 390 395 400 Ala Val Gly Pro Gly Phe Thr Gly Gly Ala Arg Gln Thr Arg Thr Ala 405 410 415 Ala Pro Val Asp Ile Pro Ala Thr Val Cys Ala Leu Leu Gly Arg Asp 420 425 430 Val Pro Asn Asp Trp Asp Gly Val Pro Leu Ser 435 440 132 123 PRT Streptomyces lavendulae 132 Met Thr Pro Glu Glu Leu Ser Asp Phe Ala Leu Glu Leu Pro Glu Ala 1 5 10 15 Val Asp Asp Glu Ala Phe Gly Pro Gly Ala Ala Val Phe Lys Val Glu 20 25 30 Lys Lys Val Phe Ala Ile Leu Gln Asp Ala Ser Glu Asp Arg Pro Pro 35 40 45 Gln Val Thr Leu Lys Cys Glu Pro Asp Leu Ala Leu His Leu Arg Glu 50 55 60 Gln Tyr Ala Ala Val Val Pro Gly Tyr His Val Asn Lys Arg His Trp 65 70 75 80 Asn Thr Val Val Leu Asn Gly Thr Val Pro Val Glu Glu Leu Arg Glu 85 90 95 Met Val Glu His Ser Tyr Asp Arg Val Val Ala Gly Leu Pro Lys Ala 100 105 110 Val Arg Glu Arg Leu Arg Leu Leu Arg Thr Val 115 120 133 351 PRT Streptomyces lavendulae 133 Met Thr Val Glu Gln Thr Pro Glu Asn Pro Gly Thr Ala Ala Arg Ala 1 5 10 15 Ala Ala Glu Glu Thr Val Asn Asp Ile Leu Gln Gly Ala Trp Lys Ala 20 25 30 Arg Ala Ile His Val Ala Val Glu Leu Gly Val Pro Glu Leu Leu Gln 35 40 45 Glu Gly Pro Arg Thr Ala Thr Ala Leu Ala Glu Ala Thr Gly Ala His 50 55 60 Glu Gln Thr Leu Arg Arg Leu Leu Arg Leu Leu Ala Thr Val Gly Val 65 70 75 80 Phe Asp Asp Leu Gly His Asp Asp Leu Phe Ala Gln Asn Ala Leu Ser 85 90 95 Ala Val Leu Leu Pro Asp Pro Ala Ser Pro Val Ala Thr Asp Ala Arg 100 105 110 Phe Gln Ala Ala Pro Trp His Trp Arg Ala Trp Glu Gln Leu Thr His 115 120 125 Ser Val Arg Thr Gly Glu Ala Ser Phe Pro Ser Thr Trp Pro Thr Ala 130 135 140 Pro Arg Ser Gly Ser Ser Pro Thr Arg Asp Pro Lys Ala Arg Glu Leu 145 150 155 160 Phe Asn Arg Ala Met Gly Ser Val Ser Leu Thr Glu Ala Gly Gln Val 165 170 175 Ala Ala Ala Tyr Asp Phe Ser Gly Ala Ala Thr Ala Val Asp Ile Gly 180 185 190 Gly Gly Arg Gly Ser Leu Met Ala Ala Val Leu Asp Ala Phe Pro Gly 195 200 205 Leu Arg Gly Thr Leu Leu Glu Arg Pro Pro Val Ala Glu Glu Ala Arg 210 215 220 Glu Leu Leu Thr Gly Arg Gly Leu Ala Asp Arg Cys Glu Ile Leu Pro 225 230 235 240 Gly Asp Phe Phe Glu Thr Ile Pro Asp Gly Ala Asp Val Tyr Leu Ile 245 250 255 Lys His Val Leu His Asp Trp Asp Asp Asp Asp Val Val Arg Ile Leu 260 265 270 Arg Arg Ile Ala Thr Ala Met Lys Pro Asp Ser Arg Leu Leu Val Ile 275 280 285 Asp Asn Leu Ile Asp Glu Arg Pro Ala Ala Ser Thr Leu Phe Val Asp 290 295 300 Leu Leu Leu Leu Val Leu Val Gly Gly Ala Glu Arg Ser Glu Ser Glu 305 310 315 320 Phe Ala Ala Leu Leu Glu Lys Ser Gly Leu Arg Val Glu Arg Ser Leu 325 330 335 Pro Cys Gly Ala Gly Pro Val Arg Ile Val Glu Ile Arg Arg Ala 340 345 350 134 546 PRT Streptomyces lavendulae 134 Met Thr Val Leu Gly Leu Gly Gly Ser Gly His Asp Trp Ala Ser Cys 1 5 10 15 Ala Thr Asp Gly Arg Arg Leu Val Ala Ile Asp Glu Glu Arg Leu Val 20 25 30 Arg Ser Lys Tyr Gly Leu Gly Ala Asp Leu Leu Ala Gly His Ser Arg 35 40 45 Arg Ala Val Leu Asp Ala Leu Gly Thr Ser Ala Glu Ala Val Glu His 50 55 60 Val Val Ala Cys Glu Leu Val Pro Arg Pro Phe Tyr His Ser Phe Arg 65 70 75 80 Arg Arg Val Thr Val Val Asn His His Leu Ala His Ala Tyr Ser Ala 85 90 95 Phe Gly Ala Ser Gly Met Thr Arg Ala Ala Val Leu Val Cys Asp Asn 100 105 110 Ser Gly Ser Leu Val Thr Gly Leu Lys Ser Gly Pro Gly Pro Arg Glu 115 120 125 Ala Glu Thr Ile Ser Cys Tyr Thr Ala Asp Ala Ser Gly Leu Arg Leu 130 135 140 Val Asn Arg Val Ala Gly Thr His Ala Val Asp Ala Ser Ser Glu Ser 145 150 155 160 Ala Tyr Tyr Gln Pro Gly Glu Thr Asp Asn Ser Leu Gly His Phe Tyr 165 170 175 Arg Ser Ala Ser Leu Ala Leu Gly Leu Ala Tyr Ser Gly Pro Lys Thr 180 185 190 Arg Tyr Pro Val Ser Glu Asp Gly Lys Thr Met Gly Leu Ala Pro Tyr 195 200 205 Gly Asp Asp Arg Phe Val Asp Glu Val Ala Glu Leu Val Thr Leu Leu 210 215 220 Pro Glu Gly Gly Val Gln Ile Ser Ala Ser Lys Val Asn His Leu Phe 225 230 235 240 Glu Arg Leu Val Glu Ser Gly Glu Phe Glu Asp Arg Ala Ala Leu Ala 245 250 255 Tyr Ala Ala Gln Glu Thr Leu Glu Arg Ala Leu Leu His Cys Ala Arg 260 265 270 Asp Leu His Arg Arg Thr Gly Leu Thr Asp Leu Cys Ile Ala Gly Gly 275 280 285 Val Gly Leu Asn Ser Val Ala Asn Gly Arg Ile Leu Arg Glu Thr Pro 290 295 300 Phe Glu Arg Val Phe Val Val Pro Ala Ala Gly Asp Asn Gly Ile Ser 305 310 315 320 Leu Gly Cys Ala Tyr Tyr Gly Leu His Glu Leu Glu Gly Arg Ala Pro 325 330 335 Ser Glu Leu Pro Ala Leu Asp Thr Ala Tyr Leu Gly Pro Asp Tyr Pro 340 345 350 Ala Glu Arg Val Asp Ala Ala Leu Ala Gly Ser Gly Phe Thr Val Glu 355 360 365 Thr Pro Asp Asp Leu Pro Gly Arg Val Ala Gly Leu Leu Ala Glu Gly 370 375 380 Lys Ile Ile Gly Trp Phe Asp Gly Arg Ser Glu Phe Gly Pro Arg Ala 385 390 395 400 Leu Gly His Arg Ser Ile Leu Ala Ala Pro Phe Pro Ala Ser Val Arg 405 410 415 Asp His Leu Asn Asp Asn Val Lys His Arg Glu Trp Phe Arg Pro Tyr 420 425 430 Ala Pro Ile Val Arg Glu Asp Arg Ala Ala Asp Tyr Phe Asp Leu Val 435 440 445 Gln Pro Ser Pro Phe Met Leu Val Val Ala Arg Val Thr Arg Gln Asp 450 455 460 Ala Ile Pro Ala Ala Thr His Val Asp Gly Thr Ala Arg Leu Gln Thr 465 470 475 480 Leu Asn Ala Ala Gln Asn Pro Lys Val Tyr Glu Leu Leu Gly Arg Phe 485 490 495 Glu Ala Leu Thr Gly Cys Ala Val Leu Leu Asn Thr Ser Phe Asn Val 500 505 510 Ala Gly Gln Pro Ile Val Glu Thr Pro Glu Asp Ala Val Glu Ala Phe 515 520 525 Ala Gly Met Arg Leu Asp His Leu Val Val Gly Asp Arg Leu Ala Thr 530 535 540 Lys Pro 545 135 568 PRT Streptomyces lavendulae 135 Met Asp Val Pro Val Leu Val Val Gly Gly Gly Pro Thr Gly Leu Ala 1 5 10 15 Met Ala Leu Phe Leu Ala Arg His Gly Val Gly Cys Leu Leu Val Glu 20 25 30 Arg Arg Thr Thr Thr Ser Pro Val Pro Arg Ala Thr His Val Ser Arg 35 40 45 Arg Ser Met Glu Leu Phe Arg Glu Ala Gly Leu Glu Glu Glu Ile Arg 50 55 60 Arg Ala Gly Phe Glu Val Val Arg Glu Asp Asp Pro Arg Leu Arg Thr 65 70 75 80 Arg Pro Glu Arg His Leu Pro Arg Val Val Leu Gln Ala Ala Ser Leu 85 90 95 Ala Gly Pro Gly Pro Val Gly Val Leu Glu Thr Gly Asp Glu Glu Leu 100 105 110 Ala Val Pro Gly Pro Cys Ala Pro Phe Trp Cys Gly Gln Asp Arg Met 115 120 125 Glu Pro Leu Leu Ala Lys Ala Ala Ala Arg His Gly Ala Asp Val Arg 130 135 140 Phe Gly His Glu Leu Thr Gly Leu Trp Pro Gly Glu Asp Ser Thr Arg 145 150 155 160 Ala Arg Val Arg Ala Ala Gly Thr Gly Arg Thr Tyr Thr Val Asp Ala 165 170 175 Arg Phe Val Ile Ala Ala Asp Gly Ala Arg Gly Glu Ile Ala Glu Arg 180 185 190 Val Gly Ile Ala Arg Glu Gly Leu Gly Thr Val Ala His Arg Val Ser 195 200 205 Ile Leu Phe Arg Ala Asp Pro Gly Arg Trp Ala Arg Asp Arg Arg Phe 210 215 220 Phe Met Cys Met Ile Gln Asn Pro Gly Phe Asp Gly Ala Val Met Glu 225 230 235 240 Leu Asn Thr Pro Gly Arg Trp Cys Ala Ala Val Asp Tyr Asp Pro Ala 245 250 255 Arg Ala Glu Pro Asp Gly Thr Tyr Ser Ala Arg Thr Cys Leu Asp Leu 260 265 270 Val Arg Ala Ala Val Gly Asp Asp Arg Ser Asp Ala Ala Val Asp Thr 275 280 285 Val Phe His Trp Lys Ala Arg His Arg Ile Ala Ala Ala Tyr Arg Ser 290 295 300 Gly Ala Val Phe Leu Ile Gly Asp Ala Ala His Leu His Pro Pro Ser 305 310 315 320 Gly Gly Tyr Gly Ser Asn Val Gly Phe Gln Asp Ala His Asn Leu Ala 325 330 335 Trp Lys Ile Ala Ala Val Leu Gly Gly Trp Ala Gly Pro Arg Leu Leu 340 345 350 Asp Thr Tyr Asp Glu Glu Arg Arg Pro Val Gly Lys Ala Thr Ala Glu 355 360 365 Gln Ser Met Leu Leu Asp Gly Val Pro Pro Glu Pro Leu Gly Gly Ser 370 375 380 Val Val Arg Cys Asp Pro Arg Thr Leu Ile Met Gly Tyr Arg Tyr His 385 390 395 400 Ser Ala Ala Val Leu Gly Pro Pro His Gly Pro Ala Phe Pro Ala Ala 405 410 415 Phe Thr Leu Arg Gly Asp Pro Gly Thr Arg Leu Pro His Val Trp Leu 420 425 430 Arg Thr Asp Ala Gly Glu Arg Val Ser Thr Leu Asp Leu Cys His Gly 435 440 445 His Phe Val Leu Leu Ser Ala Asp Pro Val Trp Ala Ala Ala Ala Ala 450 455 460 Arg Ser Ala Lys Glu Thr Gly Val Pro Leu Arg Gly His His Leu Ala 465 470 475 480 Ala Thr Gly Ser Glu Leu Ala Asp Pro Ser Gly Glu Phe Pro Arg Ser 485 490 495 Cys Gly Thr Gly Pro Ala Gly Ala Val Leu Val Arg Pro Asp Gly Met 500 505 510 Val Ala Trp Arg Thr Ala Arg Ala Val Pro Pro Asp Pro Asp Ser Ala 515 520 525 Gln Asp Leu Val Thr Ala Ala Val Arg Arg Val Leu Ala Leu Pro Glu 530 535 540 Arg Ala Ala Pro Pro Val Leu Gly Pro Pro Arg Leu Ser Arg Gly Ser 545 550 555 560 Tyr Arg Arg Val Gly Ser Asp Gly 565 136 160 PRT Streptomyces lavendulae 136 Met Lys Pro His Ser Phe Cys Thr Cys Trp Pro Gly Ala Thr Val Trp 1 5 10 15 Leu Thr Gly Pro Pro Gly Ala Gly Lys Thr Thr Ile Ala Arg Ala Leu 20 25 30 Ala Glu Arg Leu Arg Glu Arg Gly Arg Arg Val Glu Val Leu Asp Gly 35 40 45 Asp Ala Thr Arg Ala Leu Leu Thr Ala Gly Ser Ser Trp Glu Asp Arg 50 55 60 Gly Thr Gly Leu Gln Arg Val Gly Leu Met Ala Glu Val Leu Ala Arg 65 70 75 80 Asn Gly Ile Val Val Leu Val Pro Val Thr Ala Ala Arg Ala Asp Ser 85 90 95 Arg Glu Ala Val Arg Arg Arg His Glu Arg Ser Gly Thr Ala His Leu 100 105 110 Glu Val Arg Val Val Arg Asp Ala Val Pro Pro Ser Gly Leu Pro Ala 115 120 125 Pro Pro Gly Pro Asp Leu Arg Ile Ala Ala His Glu Gln Ser Ala Glu 130 135 140 Glu Ser Ala Arg Ala Leu His Arg Leu Leu Ala Glu Arg Glu Leu Ala 145 150 155 160 137 319 PRT Streptomyces lavendulae 137 Met Asn Pro Gly Arg Gly Gly Ala Tyr Ala Ala Gly Arg Asp Gly Thr 1 5 10 15 Arg Gly Thr Arg Arg Pro His Gly Leu Ser His Leu Asp Leu Leu Glu 20 25 30 Ser Glu Ser Val His Ile Phe Arg Glu Val Ala Gly Glu Phe Glu Arg 35 40 45 Pro Val Ile Leu Phe Ser Gly Gly Lys Asp Ser Ile Val Met Leu His 50 55 60 Leu Ala Leu Lys Ser Phe Ala Pro Ala Pro Val Pro Phe Ala Leu Leu 65 70 75 80 His Val Asp Thr Gly His Asn Phe Pro Glu Val Ile Ala Tyr Arg Asp 85 90 95 Arg Val Val Ala Ala Leu Gly Leu Arg Leu Glu Val Ala Ser Val Gln 100 105 110 Asp Phe Ile Asp Asn Gly Thr Leu Arg Glu Arg Pro Asp Gly Thr Arg 115 120 125 Asn Pro Leu Gln Thr Val Pro Leu Leu Asp Ala Ile Gly Arg His Arg 130 135 140 Phe Asp Ala Val Phe Gly Gly Gly Arg Arg Asp Glu Glu Lys Ala Arg 145 150 155 160 Ala Lys Glu Arg Val Phe Ser Leu Arg Asp Glu Phe Gly Gly Trp Asp 165 170 175 Pro Arg Arg Gln Arg Pro Glu Leu Trp Arg Leu Tyr Asn Gly Arg His 180 185 190 Ala Pro Gly Glu His Val Arg Val Phe Pro Leu Ser Asn Trp Thr Glu 195 200 205 Leu Asp Val Trp Gln Tyr Val Ala Arg Glu Glu Ile Glu Leu Pro Thr 210 215 220 Ile Tyr Tyr Ala His Glu Arg Glu Val Phe Arg Arg Gly Gly Met Trp 225 230 235 240 Leu Ala Pro Gly Glu Trp Gly Gly Pro Arg Glu Gly Glu Ala Val Glu 245 250 255 Lys Arg Arg Val Arg Tyr Arg Thr Val Gly Asp Met Ser Cys Thr Gly 260 265 270 Ala Val Asp Ser Ala Ala Ala Thr Val Ala Asp Val Val Ala Glu Ile 275 280 285 Ala Thr Ser Arg Leu Thr Glu Arg Gly Ala Thr Arg Ala Asp Asp Lys 290 295 300 Leu Ser Glu Ala Ala Met Glu Asp Arg Lys Arg Glu Gly Tyr Phe 305 310 315 138 163 PRT Streptomyces lavendulae 138 Met Gly Gln Asp Ser Arg Pro Arg Trp Leu Thr Asp Glu Glu Gln Arg 1 5 10 15 Val Trp Arg Gly Tyr Leu Arg Ala Thr Arg Leu Val Glu Asp His Leu 20 25 30 Asp Arg Arg Leu Gln Arg Glu Ala Asp Met Pro His Leu Tyr Tyr Gly 35 40 45 Leu Leu Val Gln Leu Ser Glu Ala Pro Arg Arg Gly Ile Arg Met Thr 50 55 60 Asp Leu Ala Arg Asn Ala Lys Ile Thr Arg Pro Arg Leu Ser His Ala 65 70 75 80 Ile Thr Arg Leu Glu Lys Leu Gly Trp Val Arg Arg Glu Ser Cys His 85 90 95 Gly Asp Arg Arg Gly Gln Asn Ala Val Leu Thr Glu Glu Gly Arg Glu 100 105 110 Val Leu Glu Lys Ser Ala Pro Gly His Val Ala Ala Val Arg Ala Ala 115 120 125 Val Phe Asp Ser Leu Thr Pro Glu Gln Val Gly Gln Leu Gly Arg Ile 130 135 140 Cys Gln Ala Ile Glu Lys Gly Leu Asp Arg Glu Gly Ala Asp Leu Pro 145 150 155 160 Trp Leu Arg 139 413 PRT Streptomyces lavendulae 139 Met Glu Arg His Asp Gly Ala Pro Gly Trp Gly Phe Thr His Thr Gln 1 5 10 15 Tyr Ser Ala Asp His Gly Glu Arg Gly Ala Thr Arg Arg Ala Gly Ala 20 25 30 Leu Leu Ser Ala Arg Pro Leu Pro Gln Asn Gln His Ile Met Gly Trp 35 40 45 Gly Ala Glu Asn Pro Glu Pro Ala Pro Gly Arg Tyr Asp Phe Glu Val 50 55 60 Leu Asp Glu Arg Val Ala Leu Met Arg Ala Thr Gly Ala Thr Pro Val 65 70 75 80 Leu Thr Leu Cys Ala Ala Pro Asp Trp Met Lys Gly Gly Arg Pro Gly 85 90 95 Arg Thr Asp Trp Ser Arg Leu Glu Thr Ala Pro Asp Pro Arg His Tyr 100 105 110 Ala Asp Phe Ala Arg Leu Ala Gly Val Ile Ala Gln Arg Tyr Pro Asp 115 120 125 Ile Arg His Phe Leu Val Trp Asn Glu Leu Lys Gly Phe Tyr Asp Glu 130 135 140 Asp Arg Arg Arg Trp Asp Tyr Glu Gly Tyr Thr Arg Leu Tyr Asn Leu 145 150 155 160 Val His Ala Glu Leu Lys Arg Arg Asn Pro Arg Asn Leu Val Gly Gly 165 170 175 Pro Tyr Ala Val Val Asp His Asp Pro Pro Ala Glu Asp Ala Ala Asp 180 185 190 Arg Ser Arg Glu Leu Arg Gly Pro Trp Gly Glu Leu Asp Gln Arg Ser 195 200 205 Ala Asp Val Ile Arg Tyr Trp Asn Ala His Lys Ala Gly Ala Asp Phe 210 215 220 Val Val Val Asp Gly Ser Ser Tyr Thr Arg Glu Gly His Arg Ala Ile 225 230 235 240 Pro Asp Glu Phe Ala Ala Thr Glu Lys Phe Ala Asp Val Thr Arg Trp 245 250 255 Val Arg Ser Val Thr Gly Leu Pro Val Trp Trp Ala Glu Trp Tyr Val 260 265 270 Glu Pro Pro Ala Glu Asp Asp Arg Pro Gly Gly Arg Asp Gly Trp Gly 275 280 285 Glu Gly His Arg Thr Ala Val Gln Ala Thr Ala Met Met Arg Leu Ala 290 295 300 Glu Ser Gly Ala Ser Ala Ala Phe Tyr Trp Asn Pro Gln Arg Thr Gly 305 310 315 320 Lys Ala Cys Pro Gly Cys Leu Trp Arg Ser Thr His Leu Arg Asp Gly 325 330 335 Gly Gly Glu Leu Pro Met Ala Gly Leu Leu Ser Arg Phe Ala Arg Glu 340 345 350 Phe Pro Pro Gly Thr Ala Phe Arg Pro Val Ala Val Thr Cys Gly Ser 355 360 365 Gly Asp Arg Val Glu Ala Leu Ala Asp Glu Ala Ala Val Leu Val Val 370 375 380 Asn Thr Glu Cys Arg Pro Val Ala Ala Arg Val Asp Gly Gln Ala Leu 385 390 395 400 Ser Leu Ala Pro Tyr Glu Val Arg Trp Leu Thr Arg Pro 405 410 140 270 PRT Streptomyces lavendulae 140 Met Glu Phe Leu Gly Pro Ala Ala Gly Val Ser Gly Ala Thr Arg Leu 1 5 10 15 Tyr Ala Val Leu Gly Asp Pro Val Ala Gln Val Lys Ala Pro Gly Leu 20 25 30 Leu Asn Pro Leu Leu Ser Glu Ser Gly Leu Asp Ala Val Val Val Pro 35 40 45 Val His Val Arg Ala Arg Asp Leu Ala Glu Val Val Glu Gly Leu Lys 50 55 60 Arg Ile Gly Asn Leu Asp Gly Leu Leu Val Thr Val Pro His Lys Ala 65 70 75 80 Ala Leu Cys Gly Leu Ala Asp Gly Leu Gly Pro Ala Ala Ala Leu Ile 85 90 95 Gly Thr Ala Asn Ala Met Arg Arg Glu Pro Asp Gly Arg Trp Tyr Ala 100 105 110 Glu Asn Phe Asp Gly Leu Gly Phe Val Gln Gly Leu Gln Ala Ala Gly 115 120 125 His Thr Val Arg Asp Arg His Val Ala Leu Val Gly Ala Gly Gly Ala 130 135 140 Gly Ser Ala Ile Ala Thr Ala Leu Leu Met Ala Asp Ala Ala Arg Val 145 150 155 160 Ser Val His Asp Thr Asp Arg Ala Gln Leu Asp Ala Leu Leu Leu Arg 165 170 175 Leu Gly Ser Arg Arg Pro Asp Gly Ile Arg Ala Leu Gly Pro Gly Asp 180 185 190 Leu Glu Ala Ala Asp Phe Ala Val Asn Ala Thr Pro Leu Gly Met Arg 195 200 205 Ser Glu Asp Pro Leu Pro Phe Asp Pro Ala Arg Val Arg Pro Asp Ala 210 215 220 Val Val Val Asp Val Val Met Lys Pro His Glu Thr Ala Leu Leu Ser 225 230 235 240 Ala Ala Ala Thr Ala Gly Arg Arg Val His His Gly Ile His Met Leu 245 250 255 Glu Gln Gln Val Pro Cys Tyr Arg Ala Phe Phe Gly Trp Pro 260 265 270 141 271 PRT Streptomyces lavendulae 141 Met Thr Arg Arg Arg Pro Thr Gly Pro Ile His Arg Arg Arg Ala Ser 1 5 10 15 Leu Thr Leu Ser Pro Thr Gly Ala Ala Met Arg Arg Asn Arg Ile Ala 20 25 30 Ala Leu Leu Pro Ala Ala Leu Ala Leu Val Gly Ile Ser Val Leu Ala 35 40 45 Pro Ala Thr Thr Ala Ser Ala Ala Ala Pro His Gly Gly Thr Ser Gln 50 55 60 Ala Ala Ala Phe Pro Val Ser Glu Ala Gln Phe Lys Gln Met Phe Pro 65 70 75 80 Lys Arg Asn Ala Phe Tyr Thr Tyr Lys Gly Leu Val Ala Ala Leu Lys 85 90 95 Ala Tyr Pro Gly Phe Ala Gly Thr Gly Ser Ala Glu Val Arg Lys Gln 100 105 110 Glu Ala Ala Ala Phe Leu Ala Asn Val Ala His Glu Thr Gly Gly Leu 115 120 125 Val Tyr Val Val Glu Gln Asn Thr Ala Asn Tyr Pro His Tyr Cys Asp 130 135 140 Arg Ser Arg Pro Tyr Gly Cys Pro Ala Gly Gln Ala Ala Tyr Tyr Gly 145 150 155 160 Arg Gly Pro Leu Gln Ile Ser Trp Asn Phe Asn Tyr Lys Ala Ala Gly 165 170 175 Asp Ala Leu Gly Ile Asp Leu Leu His Asn Pro Ser Leu Val Gln Lys 180 185 190 Asp Ala Ala Val Ser Trp Lys Thr Gly Leu Trp Tyr Trp Asn Thr Gln 195 200 205 Arg Gly Pro Gly Thr Met Thr Pro His Glu Ala Met Val Asn His Arg 210 215 220 Gly Phe Gly Gln Thr Ile Arg Ser Ile Asn Gly Ala Leu Glu Cys Asp 225 230 235 240 Gly His Asn Pro Ala Gln Val Gln Ser Arg Val Ala Asn Tyr Gln Arg 245 250 255 Phe Thr Lys Ile Leu Gly Val Ala Pro Gly Gly Asn Leu Ser Cys 260 265 270 142 391 PRT Artificial Sequence A consensus sequence 142 Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Pro Xaa Trp Pro 1 5 10 15 Gln Xaa Asp Asp Ala Glu Arg Xaa Gly Leu Xaa Arg Ala Leu Xaa Gln 20 25 30 Gly Gln Trp Trp Arg Xaa Gly Gly Xaa Glu Val Xaa Xaa Phe Glu Arg 35 40 45 Glu Phe Ala Xaa Xaa His Gly Ala Xaa His Ala Leu Ala Val Thr Asn 50 55 60 Gly Thr His Ala Leu Glu Leu Ala Leu Xaa Val Met Gly Val Gly Pro 65 70 75 80 Gly Thr Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln 85 90 95 Ala Xaa Gln Arg Leu Gly Ala Val Xaa Val Pro Val Asp Val Asp Pro 100 105 110 Xaa Thr Tyr Cys Leu Asp Xaa Xaa Ala Ala Ala Xaa Ala Val Thr Pro 115 120 125 Arg Thr Xaa Ala Ile Met Pro Val His Met Ala Gly Gln Xaa Ala Asp 130 135 140 Met Asp Ala Leu Xaa Lys Xaa Ser Ala Xaa Thr Gly Val Pro Xaa Xaa 145 150 155 160 Gln Asp Ala Ala His Ala His Gly Ala Xaa Trp Xaa Gly Xaa Arg Val 165 170 175 Gly Glu Leu Gly Ser Ile Ala Xaa Phe Ser Phe Gln Asn Gly Lys Leu 180 185 190 Met Thr Ala Gly Glu Gly Gly Ala Val Leu Phe Pro Asp Xaa Glu Xaa 195 200 205 Xaa Xaa Xaa Glu Xaa Ala Phe Leu Xaa His Ser Cys Gly Arg Pro Xaa 210 215 220 Xaa Asp Arg Xaa Tyr Phe His Xaa Thr Xaa Gly Ser Asn Xaa Arg Xaa 225 230 235 240 Asn Glu Phe Ser Ala Ser Val Leu Arg Ala Gln Leu Xaa Arg Leu Asp 245 250 255 Xaa Gln Ile Xaa Xaa Arg Xaa Glu Arg Trp Xaa Xaa Leu Ser Xaa Leu 260 265 270 Leu Ala Xaa Ile Asp Gly Val Val Pro Gln Xaa Xaa Asp Xaa Arg Xaa 275 280 285 Asp Arg Asn Xaa His Tyr Met Ala Met Phe Arg Xaa Pro Gly Xaa Thr 290 295 300 Glu Glu Arg Arg Xaa Ala Xaa Val Asp Xaa Leu Val Glu Arg Gly Xaa 305 310 315 320 Pro Ala Phe Xaa Ala Phe Arg Xaa Val Tyr Arg Thr Xaa Ala Phe Trp 325 330 335 Glu Xaa Gly Ala Pro Asp Xaa Xaa Xaa Xaa Glu Leu Ala Xaa Arg Cys 340 345 350 Pro Xaa Xaa Xaa Xaa Ile Xaa Xaa Asp Cys Xaa Trp Leu His His Arg 355 360 365 Val Leu Leu Xaa Xaa Glu Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Val Xaa 370 375 380 Ala Asp Xaa Val Xaa Xaa Xaa 385 390 143 393 DNA Streptomyces lavendulae 143 atgtcagcaa ggatttccct cttcgccgtg gtggtcgagg acatggccaa gtcgctggag 60 ttctaccgga agctgggcgt cgagatcccc gccgaggccg actccgcgcc gcacacggag 120 gccgtgctcg acggcggcat ccggctcgcc tgggacaccg tggagacggt gcgcagctac 180 gaccccgagt ggcaggcccc caccggcggc caccgcttcg ccatcgcgtt cgagttcccc 240 gacaccgcga gcgtggacaa gaagtacgcc gagctcgtcg acgccggcta cgagggccac 300 ctcaagccgt ggaacgccgt gtggggtcag cgctacgcca tcgtcaagga ccccgacggc 360 aacgtggtgg acctcttcgc gcccctcccg taa 393 144 16 PRT Artificial Sequence A motif 144 Val Xaa Gly Xaa Leu Xaa Asp Xaa Xaa Gly Arg Lys Xaa Xaa Xaa Leu 1 5 10 15 145 11 PRT Artificial Sequence A motif 145 Leu Asp Xaa Thr Val Xaa Asn Val Ala Leu Pro 1 5 10 146 23 DNA Artificial Sequence A primer 146 gctctagatc tacgtctccc gcg 23 147 26 DNA Artificial Sequence A primer 147 gcctcgagca tggacgatcc ctctcg 26 148 26 DNA Artificial Sequence A primer 148 gcctcgagcc gggaaagtga gcggca 26 149 23 DNA Artificial Sequence A primer 149 gcaagcttcg gcatcacgcg cca 23 150 26 DNA Artificial Sequence A primer 150 gcctcgagcg tcatgccgct cacttt 26 151 26 DNA Artificial Sequence A primer 151 gcctcgagta gggctcccac gggaag 26 152 30 DNA Artificial Sequence A primer 152 gggatcgcat atgccgcact ccgagctgtc 30 153 29 DNA Artificial Sequence A primer 153 gtgagccata tgacggaaac cgcgtccgc 29 

What is claimed is:
 1. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising a mitomycin biosynthetic gene cluster, a variant or a fragment thereof.
 2. The isolated and purified nucleic acid molecule of claim 1 which encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.
 3. The isolated and purified nucleic acid molecule of claim 1 which encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmncL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
 4. The isolated and purified nucleic acid molecule of claim 1 which is from Streptomyces spp.
 5. An expression cassette comprising the nucleic acid molecule of claim 1, 2 or 3 operably linked to a promoter functional in a host cell.
 6. A recombinant bacterial host cell in which at least a portion of a nucleic acid molecule comprising a mitomycin biosynthetic gene cluster is disrupted to as to result in a recombinant host cell that produces altered levels of mitomycin relative to a corresponding nonrecombinant bacterial host cell.
 7. The recombinant host cell of claim 6 in which mitomycin levels are increased.
 8. The recombinant host cell of claim 6 in which mitomycin levels are decreased.
 9. The host cell of claim 6 wherein the nucleic acid molecule which is disrupted encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.
 10. The host cell of claim 6 wherein the nucleic acid molecule which is disrupted encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
 11. A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid molecule comprising a mitomycin biosynthetic gene cluster operably linked to a promoter functional in the host cell.
 12. The recombinant host cell of claim 11 in which mitomycin levels are increased.
 13. The recombinant host cell of claim 11 in which mitomycin levels are decreased.
 14. The host cell of claim 11 wherein the genome is augmented with a nucleic acid molecule that encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.
 15. The host cell of claim 11 wherein the genome is augmented with a nucleic acid molecule that encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
 16. A recombinant host cell comprising a mitomycin biosynthetic gene cluster, the genome of which is augmented by a recombinant nucleic acid molecule, wherein the recombinant nucleic acid does not comprise a mitomycin biosynthetic gene, and wherein the recombinant host cell produces a biologically active agent that is not produced by the corresponding non-recombinant host cell.
 17. A product produced by the recombinant host cell of claim 6 or 11 which is not produced by the corresponding non-recombinant host cell.
 18. The product of claim 17 which comprises a biologically active agent.
 19. The product of claim 18 which is a mitomycin.
 20. The product of claim 18 is not a mitomycin.
 21. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes polyketide biosynthetic enzymes or a fragment thereof, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74.
 22. An isolated and purified polypeptide comprising MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
 23. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising sugar metabolism genes or a fragment thereof, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.
 24. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes an aminoDAHP synthase from Streptomyces strains that produce mitomycin.
 25. A recombinant host cell in which at least a portion of a nucleic acid sequence which encodes polyketide biosynthetic enzymes is disrupted so as to result in a recombinant host cell that produces altered polyketide levels or polyketides of altered composition relative to a corresponding nonrecombinant cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74
 26. A recombinant host cell in which at least a portion of a nucleic acid sequence which encodes sugar metabolism enzymes is disrupted so as to result in a recombinant host cell that produces altered sugar levels or molecules with altered sugar composition relative to a corresponding nonrecombinant cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.
 27. A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid sequence which encodes polyketide biosynthetic enzymes operably linked to a promoter functional in the host cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74.
 28. A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid sequence which encodes sugar metabolism enzymes operably linked to a promoter functional in the host cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.
 29. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence that hybridizes under hybridizing conditions to a nucleic acid segment comprising SEQ ID NO:96, or a fragment thereof.
 30. The isolated and purified nucleic acid molecule of claim 29 which is plant nucleic acid.
 31. The isolated and purified nucleic acid molecule of claim 29 which is prokaryotic nucleic acid.
 32. A method to introduce exogenous DNA into a refractory Streptomyces strain, comprising: a) contacting a bacterial donor cell comprising a conjugative plasmid with a Streptomyces cell so as to yield a transformed Streptomyces cell comprising at least a portion of the plasmid; and b) identifying the transformed Streptomyces cell.
 33. The method of claim 32 wherein the Streptomyces strain produces a mitomycin.
 34. A method to identify a nucleic acid molecule that is related to at least a portion of a nucleic acid molecule comprising a mitomycin gene cluster, comprising: a) contacting a sample comprising nucleic acid with an amount of a probe comprising at least a portion of a nucleic acid molecule comprising a mitomycin gene so as to form a complex; b) detecting the presence or absence of the complex.
 35. A method to identify a nucleic acid molecule that is related to at least a portion of a nucleic acid molecule comprising a mitomycin gene cluster comprising: a) contacting a sample comprising nucleic acid with at-least one oligonucleotide under conditions effective to amplify the nucleic acid so as to yield an amplification product, wherein the oligonucleotide specifically hybridizes to nucleic acid comprising a mitomycin gene cluster, and b) detecting or determining the presence or absence of the product.
 36. The method of claim 34 or 35 wherein the sample is obtained from a plant.
 37. The method of claim 34 or 35 wherein the sample is obtained from a microorganism.
 38. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising a gene product selected from MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, MmcW, MmcX, MmcY, or any combination thereof.
 39. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding at least one gene necessary for mitomycin biosynthesis.
 40. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding at least one gene for mitomycin transport.
 41. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide that regulates mitomycin biosynthesis or resistance.
 42. A method for preparing a compound or a pharmaceutically acceptable salt thereof from a recombinant host cell comprising culturing the host cell of claim 6, 11 or 16 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic fermentation conditions so as to yield an increase in the compound relative to the level of the compound produced by the corresponding non-recombinant host cell.
 43. A method for preparing a mitomycin or a pharmaceutically acceptable salt thereof from a recombinant host cell comprising culturing the host cell of claim 6, 11 or 16 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic fermentation conditions so as to yield an increase in the mitomycin relative to the level of the mitomycin produced by the corresponding non-recombinant host cell.
 44. A product produced by the recombinant host cell of claim 16 which is a mitomycin.
 45. A product produced by the recombinant host cell of claim 16 which is not a mitomycin.
 46. The product of claim 19 which is 9a-demethoxy mitomycin C. 